Glycoprotein Hormone Receptors in Sea Lamprey

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Glycoprotein hormone receptors in sea lamprey (Petromyzon marinus): Identification, characterization and implications for the evolution of the vertebrate hypothalamic-pituitary-gonadal/thyroid axes

Mihael Freamat University of New Hampshire, Durham

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Recommended Citation Freamat, Mihael, "Glycoprotein hormone receptors in sea lamprey (Petromyzon marinus): Identification, characterization and implications for the evolution of the vertebrate hypothalamic-pituitary-gonadal/ thyroid axes" (2007). Doctoral Dissertations. 395. https://scholars.unh.edu/dissertation/395

This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. GLYCOPROTEIN HORMONE RECEPTORS IN SEA

LAMPREY (PETROMYZON MARI Nil S):

IDENTIFICATION, CHARACTERIZATION

AND IMPLICATIONS FOR THE EVOLUTION OF THE VERTEBRATE

HYPOTHALAMIC-PITUITARY-GONADAL/THYROID AXES

MIHAEL FREAMAT

B.S. University of Bucharest, 1995

DISSERTATION

Submitted to the University of New Hampshire

in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

Biochemistry

September, 2007

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Dissertation Director, Dr. Stacia A. Sower Interim Associate Dean of Research of the College of Life Sciences and Agriculture, Professor of Biochemistry and Molecular Biology

£ ,LQa Dr. William R^'frumble, Professor of Biochemistry and Molecular Biology

^ , / W > ______Dr. Clyde LMDenis, Professor of Biochemistry and Molecular Biology

Dr. William A. Condon, Professor of Animal and Nutritional Sciences

^ ' A c ------Dr. William R Moyle, University of Medicine and Dentistry of New Jersey, Professor of OBGYN

c o } ~ Date

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS

I would like to thank:

My adviser, Dr. Stacia A Sower, for giving me the possibility to work in her

laboratory, for her continuous support in the lab, for her suggestions and advice

in the scientific field and in the everyday life.

Members of my Doctoral Committee: Dr. William Condon, Dr. Clyde Denis, Dr.

William Trumble, Dr. William Moyle, Dr. Thomas Kocher for their help and

understanding during my PhD stages.

I would like to express my gratitude to Dr. William Moyle for his generous help

with reagents and protocols, for the insightful discussions we had and for the

thorough comments he made on my dissertation and research in general.

All members of the Sower Lab, in particular Scott Kavanaugh for being the

greatest colleague I ever worked with. Geoff Bushold, Matt Silver, Kazu

Okamoto, Taka Kosugi, and many others, graduate and undergraduate students

with whom I have shared the lab space, the reagents, the protocols, the ups and

downs of a graduate student's life.

My wife Dana for more than I probably realize.

iii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... iii

LIST OF TABLES...... vi

LIST OF FIGURES...... vii

ABSTRACT...... ix

CHAPTER PAGE

INTRODUCTION...... 1

The Vertebrate Hypothalamic-Pituitary-Gonadal (HPG) and Hypothalamic-Pituitary- Thyroid (HPT) Endocrine A xes ...... 2

Glycoprotein Hormone Receptors (GpH-R) Structure and Function ...... 4

Evolution of Glycoprotein Hormone Receptors and Hypothalamic-Pituitary- Gonadal/Thyroid Axes ...... 11

The Sea Lamprey Petromyzon marinus and its Evolutionary Significance ...... 16

Specific A im s ...... 23

I. IDENTIFICATION AND CHARACTERIZATION OF THE LAMPREY GLYCOPROTEIN HORMONE RECEPTOR 1...... 24

Introduction ...... 24

Materials and Methods ...... 26

Results ...... 34

Discussion ...... 38

II. IDENTIFICATION AND CHARACTERIZATION OF THE LAMPREY GLYCOPROTEIN HORMONE RECEPTOR II...... 45

Introduction ...... 45

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Materials and Methods ...... 47

Results ...... 57

Discussion ...... 65

III. FUNCTIONAL DIVERGENCE OF GLYCOPROTEIN HORMONE RECEPTORS...... 69

Introduction ...... 69

Materials and Methods ...... 73

Results ...... 81

Discussion ...... 89

SUMMARY AND CONCLUSIONS...... 96

BIBLIOGRAPHY...... 97

APPENDICES ...... 114

APPENDIX A. IACUC Approval Note ...... 115

APPENDIX B. cAMP Assay Using the pCRE-SEAP Reporter System ...... 116

APPENDIX C. Oligonucleotides Used in the Study of Lamprey GpH-R I and I I ...... 122

APPENDIX D. Chemical Communication in Vertebrates ...... 123

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES

Table 1. Vertebrate glycoprotein hormone receptors molecular cloning ...... 12

Table 2. Constitutive activity of IGpH-R 1 ...... 38

Table 3. Vertebrate GpH-Rs: database records, domain boundaries and IGpH-RI and II similarities ...... 61

Table 4. Transfection reagent amounts for pCRE-SEAP/receptor co-transfection. 118

Table 5. Oligonucleotides used in the study of lamprey GpH-R I and II ...... 122

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES

Figure 1. Hypothalamic-pituitary-thyroid and hypothalamic-pituitary-gonadal axes...... 3

Figure 2. Diagram of a generic glycoprotein hormone receptor in complex with its ligand ...... 5

Figure 3. Glycoprotein hormone receptors signal transduction pathway...... 8

Figure 4. Sea lamprey life cycle ...... 17

Figure 5. Vertebrate phylogeny...... 19

Figure 6. Lamprey glycoprotein hormone beta chain ...... 21

Figure 7. The nucleotide sequence and virtual translation of IGpH-R I CDS 33

Figure 8. IGpH-R I tissue expression pattern by RT-PCR ...... 36

Figure 9. IGpH-R I cAMP functional assay...... 37

Figure 10. IGpH-R I molecular phylogeny...... 39

Figure 11. Molecular cloning of lamprey GpH-R II ...... 55

Figure 12. Lamprey GpH-R II nucleotide and protein sequences ...... 56

Figure 13. Protein fingerprints and motifs in the sequence of the lamprey GpH-R II...... 59

Figure 14. Exon/intron organization of the lamprey GpH-R I and II genes ...... 60

Figure 15. Lamprey genomic DNA Southern blot hybridization (A) and IGpH-R II tissue expression levels estimation by RT-PCR (B) ...... 62

Figure 16. Hydropathy profiles of lamprey GpH-R I, II compared with mammalian (mouse) LH-R hydropathy...... 63

Figure 17. Molecular phylogenetic analysis of glycoprotein hormone receptor protein sequences ...... 64

vii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 18. Preparation of chimeric receptors by Splicing by Overlap Extension (SOE-PCR)...... 74

Figure 19. Agarose gel of the products of SOE-PCR amplification ...... 74

Figure 20. Calculation of the SEAP initial rate ...... 78

Figure 21. Diagram of the chimeric rat LH-R/lamprey GpH-R I chimeric receptors. 80

Figure 22. Details of the rat LH-R/lamprey GpH-R I chimeras SSD level junctions. 82

Figure 23. Effect of oLH and oTSH on chimeric IGpH-R l/rLH-R receptors series A and B ...... 83

Figure 24. Analysis of constitutive activity of rat LH-R/lamprey GpH-R I chimeric receptors series C-F...... 84

Figure 25. Response of domain-swapped rat LH-R / lamprey GpH-R I chimeras (series C, lamprey ED) to oLH, hFSH, hCG and PMSG ...... 86

Figure 26. Response of domain-swapped rat LH-R / lamprey GpH-R I chimeras (series D, rat ED) to oLH, hFSH, hCG and PMSG ...... 87

Figure 27. Effect of SSD manipulation on the response of chimeras containing lamprey ED to oLH stimulation ...... 88

Figure B.28. Outline of the cAMP functional assay using the pCRE reporter system ...... 117

Figure D.29. Thyroid hormones (T3.T4) and gonadal steroid hormones ...... 126

viii

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT

GLYCOPROTEIN HORMONE RECEPTORS IN SEA LAMPREY

(PETROMYZON MARINUS): IDENTIFICATION, CHARACTERIZATION AND

IMPLICATIONS FOR THE EVOLUTION OF THE VERTEBRATE

HYPOTHALAMIC-PITUITARY-GONADAL/THYROID AXES

Mihael Freamat

University of New Hampshire, September, 2007

The vertebrate hypothalamic-pituitary-gonadal (HPG) and hypothalamic-

pituitary-thyroid (HPT) endocrine axes involve specific interaction between

pituitary glycoprotein hormones GpH (lutropin LH, follitropin FSH, thyrotropin

TSH) and glycoprotein hormone receptors GpH-R (LH-R, FSH-R and TSH-R

respectively).

GpHs and GpH-Rs originate in a common ancestral ligand/receptor pair. Their

duplications were followed by divergent evolution resulting in the emergence of a

novel, pituitary/peripheral gland control level. It is estimated that this occurred

more than 500 million years ago (mya), before or concomitant with the

divergence of the jawed vertebrates (Gnathostomes) lineage from their jawless

(Agnathan) ancestors. This coincides with the estimated time of divergence of

the lamprey lineage, one of the two surviving Agnathan groups (lamprey and

hagfishes) in the actual fauna. Therefore, the study of the GpH/GpH-R system in

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lamprey is of foremost importance for understanding the evolutionary

emergence of the HPG and HPT axes. Recently, a pituitary glycoprotein similar

with the GpH (3 chain was identified in this species.

Two glycoprotein hormone receptors (IGpH-R I and IGpH-R II of 719 and 781

residues respectively) highly similar with Gnathostome GpH-Rs were cloned from

sea lamprey testes and thyroid respectively. The highest tissue transcript levels

were found by RT-PCR in testes and thyroid respectively and their exon/intron

organization is almost identical with that of mammalian FSH-R and TSH-R.

Mammalian LH induces a low but detectable increase in cAMP concentration in

the cells transfected with IGpH-R I. Functional assays on lamprey GpH-R l/rat

LH-R domain swapped chimeric receptors suggest that IGpH-R I domains

conserved their function. Alteration of the 'hinge' or Signaling Specificity Domain

(SSD) segment of IGpH-R I by insertion/replacement with rat corresponding

fragments results in a drastic change in sensitivity to oLH which is also

dependent on domain composition of the chimera.

Therefore we hypothesize that one GpH in lamprey interacts with two GpH-Rs

in gonads and thyroid as part of a primitive HPG/T axis which antecedes the

divergence of the Gnathostome HPG and HPT systems.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. INTRODUCTION

No species will survive without selective advantage that enables it to exist and

reproduce in its environment. The observation that all complex multicellular

organisms have gropus of differentiated cells that are separated into clusters of

organs suggests that this type of specialization was essential for the evolution of

most complex life forms, including vertebrates.

Specialization also appears to have been made possible by the development

of neural and endocrine networks that enabled the function of the diverse cells in

the organism to be coordinated. Communication between cells involves the

release of specialized information carrier molecules (hormones), their transport to

the target tissue (usually in blood), and recognition, binding and activation of

specific receptors. The hormone receptors act as information transduction

molecules allowing the metabolism of the target cells to respond to the changes

in their environment. The hormone/receptor pairs of the endocrine systems of

vertebrates are coupled into chains of regulatory interactions that enable the

organism to react to internal as well as to external stimuli.

The cascade of hormonal factors of the hypothalamic-pituitary-peripheral

gland control systems are organized into a multileveled architecture. This

hierarchical organization of the hormonal systems is divided into 'endocrine axes'

corresponding largely to the specific physiological compartments affected. These

systems developed to enable neural inputs to influence endocrine processes. At

the topmost segment, the activity of the endocrine systems is controlled by the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. products of secretion of the neurons in neuro-secretory nuclei located in the

hypothalamus and pre-optic area of the brain. They are released into the

hypothalamic-pituitary portal system and bind to their cognate receptors located

in specific groups of cells in the anterior pituitary gland. Here they activate the

synthetic pathways leading to production of the hypophyseal tropic hormones.

The tropic hormones are secreted into the general circulation and reach their

target organs and tissues. In spite of the very diverse patterns of life cycles and

reproductive strategies and behaviors, these endocrine systems are remarkably

conserved throughout the Gnathostome lineage [1].

In this thesis I describe two of the earliest components of the organism that

are known to be essential to regulation of the vertebrate metabolism and

reproduction: the Hypothalamic-Pituitary-Thyroid (HPT) and Hypothalamic-

Pituitary-Gonadal (HPG) endocrine control axes.

The Vertebrate Hvpothalamic-Pituitarv-Gonadal (HPG) and Hypothalamic-

Pituitarv-Thvroid (HPT) Endocrine Axes

Hormones of the hypothalamic-pituitary-gonadal (HPG) axis are involved in

coordination of the reproductive physiology, in the development of the

reproductive organs and in the control of the life cycles in most vertebrates. The

pituitary tropic hormones of this axis, luteinizing hormone (LH) and follicle

stimulating hormone (FSH), are collectively known as gonadotropins (GtH) and

are synthesized and released into the blood stream in response to the action of a

specific hypothalamic releasing factor (gonadotropin releasing hormone, GnRH).

Once in the bloodstream they travel to the target organs (gonads) where they

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. stimulate a cascade of processes which result mainly in the synthesis of steroid

hormones. The thyroid endocrine axis is defined as the assembly of hormones

and their anatomic-functional units implied in the regulation of the synthesis of

HPT Axis Female HPG Axis Male HPG Axis

Hypothalamus Hypothalamus m Hypothalamus t© ^--- . I® GnRH | (J3nRH3

Anterior Antenor Anterior pituitary pituitary pituitary ©/© © * ( £ ) ^Thyroid .r ig t. M ^ S s r - Ovaries © Testes

(^S tio g ^ ) (^Progesterone) Testosterone

Figure 1. Hypothalamic-pituitary-thyroid and hypothalamic-pituitary-gonadal axes. Reproduced from [2].

thyroid hormones from the thyroid gland. It has a similar organization, the

thyrotropin tropic hormone being released from anterior pituitary thyrotroph cells

into the general circulation in response to the action of the hypothalamic

thyrotropin releasing hormone.

The thyroid endocrine axis is responsible primarily for regulation of cellular

energy metabolism. In different organisms, thyroid hormones are important

factors controlling various stages of development. In amphibians for example, the

thyroid hormone secretion is associated with the onset of metamorphosis.

The specificity of action of these two endocrine axes is in fact determined by

the binding affinity between the glycoprotein hormones (GpH) and their

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. receptors (GpH-R) as well as by the pattern of expression of these receptors.

The glycoprotein hormones are dimeric proteins composed of two subunits linked

by non-covalent interactions. In vertebrates one subunit is common in all

glycoprotein hormones (the alpha chain) while the second subunit is distinct so it

primarily confers specificity to the interaction with the receptor. The receptors for

glycoprotein hormones belong to the G-Protein Couple Receptors (GPCR)

superfamily of membrane receptors and are grouped into a distinct subfamily

characterized by a large extracellular domain which is half of the total length of

the mature protein. Recently a number of newly identified and characterized

mammalian receptors have been shown to share the same general molecular

organization with the GpH-Rs; therefore the family of proteins including the GpH-

Rs has been extended to include these new molecules [3].

Glycoprotein Hormone Receptors (GpH-R) Structure and Function

Three types of receptors for glycoprotein hormones i.e. the gonadotropin

receptors (follitropin receptor, FSH-R and lutropin receptor, LH/CG-R) and

thyrotropin receptors (TSH-R) have been described in vertebrates.

After the advent of the molecular biology methods of cDNA cloning, a large

number of glycoprotein hormone receptors have been identified. The first

characterized were the luteinizing hormone receptors (LH-R) from pig and sheep

ovaries [4], [5]. Most of the receptors are from mammalian species but

glycoprotein hormone receptors have also been characterized from species of

birds [6], [7], [8], reptiles [9], and (particularly after 2000) fish (reviewed in [10]).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Moreover, glycoprotein hormone receptor-like proteins have been identified in

invertebrates (sea anemone, Anthopleura elegantissima ([12]), the fruit fly,

Drosophyla melanogaster [13] and the nematode Caenorhabditis elegans [13]).

Analysis of the protein sequences obtained by virtual translation from cDNA

sequences has shown that the glycoprotein hormone receptors belong to the

GPCR. They define a sub-family of GPCRs (glycoprotein hormone receptors,

GpH-R) which features a large extracellular, hydrophilic N-terminal end

accounting for half of the total length of the molecule (700 amino acid residues in

average), a transmembrane hydrophobic region and an intracellular domain. The

p-chain a2

'seat belt' a-chain SSD\ ('hinge' Transmembrane Domain (TMD)

70000000000000

^yacellular Domain

Figure 2. Diagram of a generic glycoprotein hormone receptor in complex with its ligand. Modified from [11],

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. extracellular domain has a heterogeneous organization, containing a Leu Rich

Repeat Domain (LRD) flanked by two Cys Rich boxes [14],[15],[16], [11].

The LRD and transmembrane domains are linked by a highly divergent

intervening segment. This region is usually termed the 'hinge' region and is

assigned a simple, merely connective, functionality; however, there is strong

experimental support for the concept of an important role played by this domain

in modulation of the selectivity of the receptors towards their ligands and within

this context the linker is termed Signal Specificity Domain (SSD, [17]).

Understanding of the tridimensional structure of the glycoprotein hormone

receptors is limited by the difficulty in obtaining purified samples of the protein to

be used in X-ray crystallography This is why most of the 3D information

regarding these proteins has been been derived from structural modeling of the

Leu Rich Repeat domain (LRD) and transmembrane domain (TMD) based on

their homology with ribonuclease inhibitor and rhodopsin, respectively.

A tridimensional structure of the FSH receptor extracellular domain in complex

with its ligand has been resolved and published recently ([18]).

In mammals the tissue expression of the glycoprotein hormone receptors is

remarkably specific, with the gonadotropin receptors being expressed in gonads

and the thyrotropin receptor being expressed in follicular cells of the thyroid. The

specificity of expression decreases in the order FSH-R to TSH-R to LH-R. There

have been described cases of 'ectopic' expression of LH-R and TSH-R ([19],

[20]). However, their role in these tissues is not completely understood. It has

also been hypothesized that transcription of the LH-R or TSH-R genes in tissues

other than gonads or thyroid may not result in mature, functional receptors ([16]).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The specificity of expression of GpH-R also decreases in early evolved

vertebrates ([21], [22])

It is now known that glycoprotein hormone receptors interact with their

receptors at the level of the extracellular domain ([14], [15], [11]). The details of

the binding and signal transduction activation as well as the relative role of each

of the extracellular domain segments in selectivity of the receptor in respect to

different glycoprotein hormone ligands are still debated and subject to active

investigation ([23], [17]).

In terms of receptor selectivity, there is cross binding between gonadotropins

and their receptors which goes beyond species -- for example human chorionic

gonadotropin (hCG) is regularly used to probe activation of the signal

transduction pathways by novel putative gonadotropin receptors usually in

mammalian in vitro expression systems. Moreover, there is a cross binding

between glycoprotein hormones: the binding of human CG to human thyrotropin

and follitropin receptors has been studied using chimeric (“yoked”) proteins

hCG/hTSHR, hCG/hLHR, hCG/hFSHR; it has been concluded that the

interaction between hCG and TSHR may be important in some physiological

situations (early pregnancy) [24]. Conversely, the binding of thyrotropin hormone

to the FSH-R has been confirmed by in vitro experiments as a possible cause for

the effects of hypothyroidism on testicular development in humans ([25]).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FSH

Ca 2+

I IISM&ISSsS.umthvv.'. j t . k calcium channel GTPI:1:^5:i!n5Li«--Lr / R|F

protein kinase A P © » | | —► [^"1 protein hosphorylation transcrip ©

<5 mRNA scription nucleus

Figure 3. Glycoprotein hormone receptors signal transduction pathway. The figure (reproduced from reference [15]) shows the signal transduction pathway of the follicle stimulating hormone receptor FSH-R.

The glycoprotein hormone receptors have been shown to act through

activation of the cAMP dependent signal transduction pathways. The ligand

interacts with the receptor at the level of the extracellular domain and induces the

signal transduction through a cAMP/adenylate cyclase pathway activating the

steroidogenesis and secretion of testosterone, estradiol or progesterone; an

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. alternate pathway activated by hormone binding might in some circumstances be

the PLC/IP3 signaling pathway [26].

The genes encoding the gonadotropin hormone receptors have 11 exons and

10 introns (LH-R) or 10 exons and 9 introns (FSH-R ) ([14], [15], [11]).

Remarkable is the fact that in vertebrates all introns are localized in the

extracellular domain, with the only exception (so far) being the salmon

gonadotropin receptors which have an intron in the transmembrane domain [21]).

Analysis of the gonadotropin receptor transcripts by Northern hybridization or

primer extension assays has detected multiple species of RNA; the possible

mechanisms responsible for generation of these alternate transcripts include the

existence of multiple transcription starting sites, alternate polyadenylation sites,

and alternative splicing of the primary transcript (reviewed in [16] and [27]).

It has been suggested that the various species of alternatively spliced

receptors have a role in modulating the activity of the full variant but this

hypothesis has not been confirmed experimentally [28]. The systematic study of

the regulatory regions of the gene has been done for mammalian GTH-Rs. The

promoter responsible for the basal expression of the LH receptor in the rat has

been identified in a region encompassed by the translation start site and position

-173 at the 5' region of the receptor [29]. The gene lacks TATA or CCAAT

promoter elements but in the same region there have been multiple binding sites

identified for SP1 promoter specific transcription factors and also multiple

putative transcription starting sites. The region between -174 and -990 may

contain inhibitory elements that are considered responsible for the differential

regulation of the expression of the receptor. The region beyond position -991 is

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. also considered to contain inhibitory elements responsible for the absence of

expression of the gene in certain cell lines [30], [31], [32].

The FSH-R gene promoter in mammals (rat and human) has been assigned to

the region between position -1 and -286; it also lacks TATA or CCAAT elements

and shows multiple transcription starting sites, but it doesn't contain the SP1

binding sites found in LH-R promoters. A putative cAMP Response Element

(CRE) has been identified in the rat FSH-R promoter but not in the human or

mouse [15]. On the other hand, the human FSH-R promoter features a putative

estrogen responsive element which is not present in the rat FSH-R gene

promoter. Similar with the LH-R, the 5' region upstream of the putative promoter

region may contain cis-acting repressor elements. Both LH-R and FSH-R in

mammals are constitutively expressed in cell lines ( see [33] and references

therein). All these data indicate a regulation of the GTH-R gene transcription

which is both species and receptor specific.

In vertebrates, the functional specificity of the glycoprotein hormones, lutropin,

follitropin and thyrotropin depends on their specific and time controlled

expression in a few specialized tissues. Deviations from these patterns of action

are usually associated with pathological conditions. This imposes highly

specialized gene expression regulation mechanisms for each receptor. The

specificity in the expression of these receptors together with the increase in the

affinity of binding of the cognate ligand are two evolutionarily refined pathways

towards the increase in the complexity and modularity of these endocrine axes.

The expression distribution pattern shows a sharper differentiation between

species: in mammalian species the distribution of the expression of the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. glycoprotein hormone receptors seems more tissue specific than in non

mammalian vertebrates. Although there have been reports of “ectopic”

expression of the LH/CG receptor (e.g. in human brain [19], human sperm [34] or

even human thyroid [35]) and of the TSH receptor (e.g. in rat fat cells [36] or

anterior pituitary [37]) in mammals), the tissue distribution seems much more

specific than the situation found for example in catfish for TSH-R [22] or for LH-R

[38]. In contrast, the tissue distribution of the follicle stimulating hormone receptor

(FSH-R) is basically restricted to the gonads in all vertebrate species [15],

including fish [21].

Evolution of Glycoprotein Hormone Receptors and Hvpothalamic-Pituitarv-

Gonadal/Thvroid Axes

The Origins and Evolution of Chemical Communication.

Animal hormones have their precursors in the ancestral unicellular organisms.

The precursors for the key signaling molecules predate the animal origins

(reviewed in [39], [40], [41]). Also, some of the main forms of receptors have

identifiable possible precursors in the unicellular organisms. Examples are the G-

protein coupled receptors involved in the glucose sensitivity in yeasts ([42]) and

the membrane receptors for insulin like factors in Tetrahymena or yeasts ([43],

[44]). These primitive elements of chemical communication in unicellular

organisms seem to be involved in nutrient related chemotaxis and proto-sexual

pheromone like communication between individual cells ([45]). In the conclusive

remarks to a comprehensive molecular phylogenetic study of the members of

the nuclear receptor superfamily, Laudet ([46]) suggests that this class of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. receptors is only characteristic of Metazoans, same as the hox group of

homeodomain proteins. However, proteins (’receptors') capable of binding

hormone like steroid ligands (e.g. a dihydroepiandrosterone (DHEA) binding

protein) as well as metabolic effects of steroid treatments in the ciliate

Tetrahymena have been described ([47], [48])

The iodothyronine (thyroid hormones) roles in the regulation of basal

metabolism might have originated in the products of iodine metabolism in

primitive marine animals feeding on iodine-rich algae [49], [50].

Molecular Evolution of Pituitary Glycoprotein Hormones and their Receptors.

Table 1. Vertebrate glycoprotein hormone receptors molecular cloning.

Receptor Organism Tissue Method ID

LH-R Sus Scrofa (pig) gonads gt11 expression library screening Loosfelt 1989

LH-R Rattus rattus (rat) ovary cDNA library screening McFarland 1989

LH-R Homo sapiens (human) cDNA library screening Minegishi 1990

FSH-R Rattus rattus (rat) testis Sertoli cells cDNA library screening Sprengel 1990

FSH-R Homo sapiens (human) cDNA library screening [51]

FSH-R Homo sapiens (human) testis testis cDNA library Kelton 1992

GTH-R like Anthopleura elegantissima (sea anemone) cDNA library screening [121

FSH-R Macaca fascicularis (crab eating testis RT-PCR Gromoll 1993 macaque)

FSH-R Equus cabalius (horse) testis RT-PCR followed by RACE Robert 1994

LH-R Sus Scrofa (pig) testis Monoclonal antibodies raised against porcine LH Misrahi 1994 receptor and allowed to clone the corresponding cDNA from testicular cells.

FSH-R Sus Scrofa (pig) ovary RT-PCR followed by RACE Remy 1995

FSH-R Gallus gallus (chicken) ovary RT-PCR followed by RACE You 1996

LH-R Galius gallus (chicken) ovary RT-PCR followed by library screening Johnson 1996

FSH-R Ovis aries (sheep) testis RT-PCR followed by PCR screening of a cDNA library Yamey 1997

FSH-R Gallus gallus (chicken) ovary RT-PCR followed by cDNA library screening Wakabayashi 1997

LH-R Callithrix jacchus (common marmoset) testis RT-PCR followed by RACE [52]

FSH-R Equus asinus (donkey) testis RT-PCR Richard 1997

GTH-R-like (fly Drosophila melanogaster (fruit fly) cDNA library screening [13] LGR1)

LH-R Gallus gallus (chicken) ovary, granulosa cDNA library screening Mizutani 1998 cells

GTH-R II Oncorhynchus rhodurus (amago salmon) ovarian follicles O ba1999

GTH-R 1 Oncorhynchus rhodurus (amago salmon) ovarian follicles O b a 1999

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1 cont nued GTH-R like (fly Drosophila melanogaster (fruit fly) RT-PCR followed by library screening [53] LGR2)

FSH-R Cynops pyrrhogaster (japanese fire belly testis Nakayama 2000 newt)

GTH-R. partial Labeo rohita (rohu fish) PCR, genomic DNA Kumaresan 2000

GTH-R like Caenorhabditis elegans PCR [54] (LGR)

LH-R Meleagris gallopavo (wild turkey) ovary RT-PCR followed by RACE [8]

FSH-R Podards sicula (lizard) ovary Borrelli 2001

FSH-R Ictalums punctatus (channel catfish) gonads RT-PCR followed by RACE [10]

LH-R Ictalums punctatus (channel catfish) gonads RT-PCR followed by RACE [10]

FSH-R Clarias gariepinus (african catfish) testis RT-PCR followed by library screening [55]

GTH-RI Oreochromis niloticus (Nile tilapia) [21]

GTH-R II Oreochromis niloticus (Nib tilapia) [21]

FSH-R Macropus eugenii (tammar walbby) ovary Mattiske 2002

G TH -R 1 Danb redo (zebrafish) ovary Laan 2002

LH-R Clanas garbpinus (african catfish) gonads RT-PCR followed by library screening [22]

Recently a number of putative receptors with similar molecular organization

have been characterized in invertebrate species as well as in mammals. This

group of receptors, featuring a large, Leu rich repeat containing extracellular

domain, extended the GpH-R subfamily, which is now considered as part of a

larger group - the LGR (Leu Rich Repeat Containing GPCR) receptors ([3]).

The glycoprotein hormone receptors have hypothetically evolved by

duplication of a common ancestor resulted from recombination of a GPCR and

an LRR protein [3]. One mechanism of evolutionary functional divergence of

glycoprotein hormone receptor paralogs is the variation in protein sequence

(coding region of the gene). The correlated change in receptor and hormone

binding site sequences (receptor/ligand co-evolution) is one explanation

proposed for the increased ligand binding specificity [56]. Another hypothesis is

that the binding specificity evolved independently from signal transduction by

introduction of domains (“negative-specificity” domains) that block inappropriate

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ligand-receptor interactions and decrease the affinity of binding of non cognate

ligands [57]. The binding affinities reflect specific physiological requirements; for

example the mouse and bovine TSH have higher intrinsic activity than human

TSH [57]. Considerably less attention has been devoted to other mechanisms of

evolution such as the change in tissue-specific regulation of the receptor gene

expression [58]. It is accepted that one pathway of increasing developmental

complexity is by partitioning the roles of the duplicated genes by changes in their

expression regulation; for example, through differential silencing of the

enhancers [59].

It has also been shown ([60]) that genes responsible for physiological traits

can exhibit shifts in expression mapped to the 5' regulatory region even between

populations of the same species. The effect of the evolutionary change in the

regulatory pattern of a gene in a species is dependent on its position in the

hierarchical network of regulatory interactions which control the developmental

processes in that species ([58]).

The evolution of proteins took place through a chain of intermediates

exhibiting small, incremental changes, associated with gradual increase in

fitness. This minimizes the role of the blind genetic drift in evolution of protein

functions [61].

Conserved scaffolds and active sites are crossover points for recombination

between members of the same family of proteins resulting in new, related

proteins with new functions [62]. The path to an increased fitness of the protein

may go through necessary steps, some of which are in fact deleterious for the

protein, and can be associated with down-slopes on the fitness landscape.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. These steps might be counterbalanced by subsequent (epistatic) changes which

actually increase the fitness of the protein to a level higher than the one

attainable through a regular all-rising evolutionary pathway [61].

Interacting proteins evolve at similar rates [63]. Their interaction imposes

reciprocal structural constraints upon each other resulting in a similar rate of

evolution between the interacting pair. Calculation of the mean difference in

fitness effects between interacting proteins resulted in significantly lower values

when compared with a similar set calculated for a randomized collection of

protein pairs selected from yeast and Caenorhabditis elegans genomes [63]. This

suggests that the similar effects on organism fitness of both members of the

interacting pair may determine their similar evolutionary rates. However, causal

path analysis showed that the correlation between the fitness effects explains

only marginally the correlation between their evolutionary rates [63]. The authors

conclude that the main factor contributing to the similar evolutionary behavior of

the interacting proteins is not necessarily their importance for the organism but

merely the structural constraints imposed by their interaction. This concept is

further investigated by Hakes et al. [64] and the distinction between co-evolution

(due to physical interaction of proteins) and correlated evolution (due to their

functional relatedness) is explicitly stated.

A still unsolved question of molecular evolution is how the hormone receptor

lock and key interaction models function in the context of the evolution of the

hormone receptor complexes under a Darwinian perspective. It presumes the

pre-existence of a key so that the receptor will evolve towards a form in which it

best fits this interaction. The problem of the evolution of the receptor/ligand pairs

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. or of the lock and key systems lies right at the basis of the whole evolutionary

theory [65]. These pairs simply can not evolve. The evolutionary relationship

between the glycoprotein hormones and their receptors is even more complex

since both partners are capable of evolution by small changes in their sequence

(Darwin's gradual change). Looking only at part of the picture, that is at the

relationship between the ligands and receptor from only one major phylogenetic

lineage, may obscure the actual path by which the receptor/ligand complexes

have actually evolved, the 'actual path of evolution'. Evolution of two traits at the

same time (the key and the lock, the ligand and the receptor) seems highly

unlikely [65], [66]. Highly deleterious mutations are rescued by subsequent

mutations which confer on the protein an advantage by inducing favorable traits,

given the context (epistatic interactions). Evolution of a protein is subject to

constraints that limit the paths through which the protein can reach a certain

specialization stage [67], [61], [68].

An alternate pathway of evolutionary change of protein functional parameters

in a physiological context is the change in the regulatory pattern of the

expression of its gene. This is able to generate major morphological changes

even in the absence of a radical modification of the protein sequence [58].

The Sea Lamprev Petromvzon marinus and its Evolutionary Significance

The sea lamprey Petromyzon marinus is a member of the Petromyzonidae,

one of the only two extant jawless vertebrates in the actual fauna. The other

agnathan representative group is the hagfishes ( Myxinidae ) now considered a

sister group of lampreys(). There are 40 species of lamprey classified in three

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. major groups. The first one, by far the most numerous includes the northern

hemisphere lampreys ( Petromyzontiformes ) while the second and third groups

are represented by the southern hemisphere lampreys (Geotria and Mordacia).

•dull tampnv iMKMd tOhW»*fi*h

nwUiMMphofU andmtoralbn to lake or «m

Figure 4. Sea lamprey life cycle.

Depending on their life cycle characteristics the lampreys are further classified

as parasitic and non-parasitic. The first category includes the species in which

one life stage includes a period when the animal attaches itself to and feeds on

the blood and bodily fluids of other fishes. In the non-parasitic species the

animals usually stop feeding following metamorphosis, and enter directly into the

final reproduction stage.

Petromyzon marinus is a northern hemisphere species, and is the largest

representative of its group and of actual jaw-less vertebrates in general. It is a

parasitic species, with a particularly complex life cycle.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. From the fertilized eggs deposited upstream in the coastal rivers hatches an

eyeless, primitively organized larva. The larval lampreys were so different from

the adult that for a long period of time the larval lampreys were classified as a

different species ( ammocoetes ). The larva lives in the river stream for a variable

number of years, ranging between 3 and 7, burrowed in the river bed and filter

feeding on the organic residues in the water. After this period (which is usually

the longest in the lamprey life cycle) it goes through a dramatic metamorphosis to

become a young adult. At this stage the animal migrates downstream into the

sea where it starts its parasitic live stage which lasts for ca. 2 years. At the end of

the parasitic phase, the reproductively mature adults migrate back up the rivers

where they originated and where they reproduce and die [69], [70].

The phylogenetic relationship between lamprey and Gnathostomes (jawed

vertebrates) is not yet established. The debate still persists particularly in respect

to the monophyly or paraphyly of the lamprey and Gnathostome group. In fact,

the problem originates in the unclear phylogenetic relationship between the

lamprey and the hagfishes. If lamprey and hagfishes belong to the same clade as

their morphological traits suggest, then lamprey are paraphyletic in respect to

Gnathostome [71]. On the other hand, some molecular phylogenetic data

indicate that lamprey are closer to jawed vertebrates than to hagfishes which

supports the monophyletic phylogeny hypothesis of lamprey and Gnathostomes

[72]. The latest lamprey fossil, Priscomyzon riniensis, a lamprey fossil discovered

in 2006 in South Africa [73] shows that the body plan of the animal was

surprisingly similar with the characteristics of the actual species. This is the

oldest fossil found to date. It is remarkably well preserved for a type of organism

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Cephalochorda

Myxinoidea (hagflsh)

Petromyzontia (lamprey)

Selachimorpha (sharks)

Batoidimorpha (rays) _ _ Chondrichbhyes Brachiopterygii (bichir) "

Chondrostel (sturgeon)

Lepisosteus (gars) Atfinopterygii

Amia (bowfin)

Teleostei (fish) Actinistia (coelocanths)

Dipnoi (lungfish)

Gymnophiona (caedlians)

Urodela (salamander)

Anura (frogs) , Amphtoia Monotremata (platypus)'

# „ # Marsupalia (marsupials) Mammalia Eutheria (placentals) , Testudinata (turtles)

Sphenodontia (tuatara)

Lizards Lepidosauria I Amphisbaenia

I Ophidia (snakes) t Crocodylia (crocodiles)

s Aves (birds) si1

Figure 5. Vertebrate phylogeny.

known not to leave many traces of its existence in the fossil deposits due to the

lack of a mineralized skeleton. The morphology of the mouth is very similar with

the oral apparatus of the actual parasitic lampreys, which suggests that they kept

their adaptive features for this style of life for over 300 million years [73].

This finding classifies the lamprey as a 'living fossil1. Until then, the lamprey

was considered a lineage which branched off the plachoderm ancestors of

vertebrates and which lost the exoskeleton during the evolution towards a

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. parasitic life style. It was also suggested an evolutionary scheme which changes

the perspective on the early evolutionary events leading to divergence of

vertebrates, the phylogeny of this animal group [73], [71].

Accordingly to the punctuated equilibrium theory, due to the low number of

speciation events between the divergence of lamprey and the contemporary

representatives of the lineage the modification in gene sequence was the result

of a gradual genetic drift, with few explosive, rapidly changing mutational bursts

associated with the speciation [74]

Understanding the hormonal control of the reproduction in Agnathans and in

particular in the sea lamprey is of foremost importance for knowing the earliest

events in the evolution of the vertebrates [72]. During the last few years, many of

the lamprey hormones have been isolated and characterized ([69], [75], [76]).

Studies done on sea lamprey reproduction have also provided evidence for the

similarity between the neuro-endocrine control of reproduction in this Agnathan

and the rest of the vertebrates. Two forms of gonadotropin hormone releasing

hormone (GnRH) have been isolated from lamprey brain (lamprey GnRH-l [77],

[78], and lamprey GnRH-lll [79]). Both GnRHs have been shown to be

hypothalamic neurohormones controlling the pituitary-gonadal axis by extensive

physiological, anatomical, biochemical and immunological studies [80],[81],[82].

A receptor for GnRH has also been isolated from sea lamprey pituitary, its

sequence determined and the functional properties of this protein were

investigated [83], [84]. Numerous efforts have also been directed towards

isolation and characterization of the pituitary hormones directly controlling the

physiology of the gonads in sea lamprey: the gonadotropin hormone homologs.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Until recently, the experimental techniques employed proved to be elusive in

isolating a gonadotropin-like hormone. There is substantial immunological and

physiological evidence that supports the role of a gonadotropin-like factor in the

lamprey pituitary. As early as 1965, Larsen showed that the effects of

hypophysectomy on gonadal function in the river lamprey (Lampetra fluviatilis)

are attenuated by the administration of lamprey pituitary extract or mammalian

gonadotropin, which indicated a relationship between the physiology of the

gonads and the pituitary hormone secretion [85]. Later it was shown that salmon

gonadotropin injections increased the level of estradiol in the reproductively

mature sea lamprey [86]. Immunocytochemical studies have shown

immunoreactive GTH in the pituitary of the sea lamprey [87]. A cDNA of a

lamprey gonadotropin has recently been cloned from the lamprey pituitary ([76]).

A Percent residue identity of the lamprey GTH beta chain with four Gnathostome LH beta protein sequences: bovine LH beta: 37% newt LH beta: 49% carp LH beta: 51 % shark LH beta: 49 %

C C C C C C C C C B m m m I ( p M * mmmmm mmmmmmm SIGNAL PEPTIDE j MATURE CHAIN

C L _LA&Jj AMLj l .

Figure 6. Lamprey glycoprotein hormone beta chain [76]. A. Identity scores of lamprey putative GTH (3 relative to selected Gnathostome gonadotropin hormone p subunits. B. diagram of the peptide sequence showing the relative positions of the signal peptide and of the 12 Cys residues in lamprey GTH p sequence. C. the degree of conservation of Gnathostome GTH p residues represented by the height of the vertical bars.

The specialized organs, tissues and molecules involved in the bioregulatory

mechanisms of the ancestral organisms have left no records in the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. paleonthological sources. Their organization and function has to be deduced

based on the more lasting remains of these animals and by comparison of their

descendants in the actual fauna. This is why species like the lamprey, which

conserve many of the characteristics of the long time extinct groups are of such

high importance for understanding of the beginnings and the mechanisms

leading to definition of the modern chemical control systems.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Specific Aims

The work described here is part of a larger project which is being performed in

the Sower Lab in the Department of Biochemistry and Molecular Biology at the

University of New Hampshire. One of the major goals of this laboratory is to

understand and delineate the complex neuro/endocrine axis of reproduction in

one of the few Agnatha species remnant in the actual fauna the sea lamprey,

Petromyzon marinus.

The work presented in this thesis addresses the pituitary/gonadal level of

endocrine control in this species, having as goals:

1. To identify and clone the receptors for glycoprotein hormones

from sea lamprey tissues

2. To determine their evolutionary and functional characteristics,

relative to Gnathostome GpH-R subfamily.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER I

IDENTIFICATION AND CHARACTERIZATION OF THE LAMPREY GLYCOPROTEIN HORMONE RECEPTOR I

Introduction

Gonadotropin and thyrotropin hormone actions are mediated through a

subfamily of G-protein coupled receptors (GPCR) namely the glycoprotein

hormone receptors (GpH-R) [88]. Known GpH-Rs share a number of unique

features. They are composed of two functionally distinct modules of similar size:

an extracellular N-terminal domain followed by a prototypical GPCR segment.

The extracellular N-terminal domain is primarily responsible for high-affinity

hormone binding and contains a central portion of nine leucine rich repeat (LRR)

motifs, flanked by N-and C-terminal cysteine-rich clusters. The C-terminal half of

the receptor contains a transmembrane region with the typical seven

hydrophobic transmembrane alpha-helices, connected by intracellular and

extracellular loops and an intracellular C-terminal domain ([14], [89], [57], [17]).

To date, approximately 79 GpHRs have been identified and described in 36

different species, mostly in mammals but also in three species of birds, two

species of reptiles, one amphibian and 10 species of fish (Hovergen database,

http://pbil.univ-lyon1.fr). There have been no GpH-Rs described in any species of

agnathans.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The glycoprotein hormone family consists of two gonadotropin hormones

(GTHs), luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and

one thyroid stimulating hormone (TSH). They are part of the cystine-knot family

of growth factors with two non-covalently bound subunits, alpha and beta. The

alpha subunit is common within a single species. The beta subunits are

homologous and convey hormone specificity ([90], [11], [91]). Therefore, these

glycoprotein hormones are believed to have evolved from a common ancestral

molecule through duplication of beta-subunit genes and subsequent divergence

([92], [56]). Two GTHs have been identified in all taxonomic groups of

Gnathostomes, including actinopterygians ([93], [94]), sarcopterygians [95], and

chondrichthyans [96], and only recently in jawless vertebrates (agnathans) [76].

Lampreys (Petromyzontidae), along with hagfish (Myxinae), are the only living

representatives of the Agnathans, the most ancient class of vertebrates, whose

lineage dates back over 530 million years [72]. Although the problem of the

evolutionary relationship between these two surviving groups of Agnathans has

not yet come to a definitive conclusion, it is well accepted that they form a

monophyletic group to the rest of the vertebrates - the Gnathostomes. Lamprey

and Gnathostomes share the same jawless ancestors and this is of foremost

importance for the contribution of the study of lamprey in understanding the

evolutionary mechanisms which led to the definition of the functional architecture

of endocrine control in vertebrates. This is particularly true for the pituitary

GpH/GpH-R pairs since it is estimated that the structural and functional

divergence of GpH-Rs coincide with the geological time of divergence of the

Gnathostomes from their jawless ancestors.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lampreys, which express two forms of GnRH, lamprey GnRH-l and lamprey

GnRH-lll [75], are important to our understanding of the reproductive success of

the first vertebrates and are likely to have retained key characteristics of the

ancestral GTH and GTH-R(s) from which modern GTH and GTH-Rs arose.

Although the lamprey GnRH-l and III with a known role in the hypothalamic-

pituitary-gonadal axis of this animal have been well demonstrated ([81], [97],

[82]), the lamprey homologs of Gnathostome GTHs, TSHs and their receptors

have eluded numerous attempts at identification. We report here the identification

of a functional lamprey glycoprotein hormone receptor (IGpH-R I) similar with

Gnathostome GTH-Rs, exhibiting its highest level of expression in the testis of

the lamprey.

Methods and Materials

Animals. Fifty reproductively mature sea lampreys were collected from the

Cocheco River fish ladder in Dover, NH during their upstream migration in May.

They were transported to the Anadromous Fish and Aquatic Invertebrate

Research (AFAIR) Laboratory of the University of New Hampshire and

maintained under continuous flow of river water and aeration at ambient

temperature until sampled. Sea lamprey tissue samples were collected at the

UNH AFAIR laboratory. Approximately 100 mg tissue fragments were flash frozen

in liquid nitrogen then kept on dry ice until stored at -80 °C.

Degenerate primers design. Degenerate primers were designed using the

PRINTS database (http://umber.sbs.man.ac.uk/dbbrowser/PRINTSA

GLYCHORMONER protein fingerprint alignment blocks as input. The PRINTS

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. database contains protein fingerprints derived by extensive statistical analysis of

protein alignments for identification of the alignment blocks (fingerprint elements)

which uniquely identify the corresponding protein family ([98], [99]). Selection of

primers was based on a simple optimality score calculated using the following

formula: S=sum(Sj)/D, where S is the final optimality score for a given degenerate

primer candidate, Sj are scores assigned to each of the properties of the

individual oligos corresponding to the degenerate primer candidate and D is the

degeneracy of the primer.

RNA isolation and degenerate PCR amplification. Total RNA of tissues was

extracted using the TRI-Reagent (MRC) method following the manufacturer's

protocol. Single strand cDNA was synthesized from ca. 1 pg testis total RNA

using the AMV reverse transcriptase and the Not-I poly-A anchored reverse

primer from Amersham (GE Healthcare, Piscataway, NJ). Amplification with

degenerate primers (IDT, Coralville I A) was done with 1.25 U Taq Polymerase

(Promega Corp., Madison Wl) in 25 pL reaction volume containing 200 nM dNTP,

3 mM MgC^, 0.5 pL GC-Melt (Clontech, Mountain View CA) and 0.4 pM of each

primer under the following cycling conditions: (1) 30 min initial incubation at 65 °C

(2) initial denaturation 5 min/95 °C and (3) 40 cycles with 1 min/94 °C

denaturation, 30 sec/65 °C annealing, 30 sec/72 °C extension. The PCR

products were separated on NuSieve(r) low melting temperature agarose gel

(FMC BioProducts, Rockland ME) and excision of DNA bands was followed by

in-gel ligation into the pGEM-T Easy vector (Clontech). The ligation product was

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. then used for transformation of JM109 Highly Competent cells (Clontech) which

were plated on LB plates treated with IPTG/X-Gal for blue/white colony

screening. Positive colonies were grown overnight in LB medium with ampicillin.

Plasmid DNA was isolated using the Wizard(r) DNA Purification System

(Promega) and sequenced at the Sequencing Service of the University of Utah.

At least two samples resulting from different amplification experiments were

sequenced for each amplicon.

RT-PCR assay. Gene specific primers derived from the transmembrane

region of the putative lamprey GpH-R identified by degenerate PCR were used

for a semiquantitative estimation of the expression of the corresponding genes in

various lamprey tissues (brain, heart, intestine, kidney, liver, muscle, testes and

thyroid) by RT-PCR. Total RNA was extracted using the TRI-Reagent (MRC,

Cincinnati OH ) method as previously described. The amount and quality of RNA

was estimated by optical density measurements at 260 and 280 nm in 10 mM

Tris buffer (pH=8.0) and by non-denaturing agarose gel electrophoresis. Ten pg

total RNA samples were incubated with 1 IU RQ1 DNase (Promega) in PCR

Buffer for 2 hours at 37 °C to remove any contaminating genomic DNA. Aliquots

of the DNase treated total RNA were run on a non-denaturing agarose gel and

the concentration of RNA for PCR reaction was normalized based on the relative

intensity of the 28S ribosomal RNA bands. An alternate method for normalization

of the total RNA amount has been employed by spotting equal volumes (1 pL) of

the DNase treated total RNA on a agarose gel. The integrated optical density of

each spot was calculated after background subtraction (NIH/lmageJ application)

and the obtained values were then used for correction of the concentrations of

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the total RNA samples used as templates in RT-PCR. The one-tube

AccessQuick(tm) RT-PCR system (Promega) was used for amplification of a 377

bp fragment from the TM region of IGpH-R I (primers C2P6f/C2P2r). A no-reverse

transcriptase control reaction was run for every sample. The PCR program was:

initial denaturation 5 min/95 °C; denaturation 40 sec/94 °C, annealing 30 sec/63

°C, extension 45 sec/72 °C for 40 cycles; final extension step 5 min at 72 °C.

Cloning of full length IGd H-R I cDNA. Ten pg lamprey testes total RNA was

reverse transcribed at 55 °C for 1 hour with Superscript lll(r) reverse

transcriptase (Invitrogen, Carlsbad CA) in the presence of the CDS III primer and

SMART IV template-switch oligo (Clontech) followed by a 15 min incubation at

the same temperature in the presence of 5 mM MnCI2 to enhance the template

switch extension of cDNA. The reaction product was then treated with RNAse H

to release the single strand cDNA from the cDNA-RNA heteroduplexes. The 5'

end of the IGpH-R I transcript was cloned by SO-RACE while the 3' end was

amplified using a gene specific forward primer and the CDS III primer (Clontech).

The step-out RACE (SO-RACE) used a modified version of the two amplification

stages protocol described in Matz et al. [100]. The PCR products which agarose

gel migration pattern matched the one predicted from the positioning of the gene

specific primers were isolated, cloned and sequenced as described previously.

The 5' and 3' fragments were assembled using the pregap4 and gap4 programs

from the Staden Package.

COS-7 expression construct Gene specific primers (IGpH-R l_TOPOf and

IGpH-R l_TOPOr respectively) containing the START and STOP codons were

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. designed and used for cloning of the full coding sequence of IGpH-R I. Briefly,

lamprey testes total RNA was reverse transcribed with Superscript III as

previously described and the cDNA used for PCR amplification of IGpH-R I full

coding sequence with Phusion(tm) High-Fidelity DNA Polymerase

(Finnzymes/NEB Ipswich, MA). The PCR product was ligated into the pcDNA(tm)

3.1/V5-His-TOPO(r) vector (Invitrogen), cloned in TOP10 competent cells and

checked by sequencing using vector specific sequencing primers as well as

gene specific primers to ensure full coverage of the IGpH-R I coding sequence

(cds). The EndoFree(r) Plasmid Maxi Kit (Qiagen, Valencia CA) was used for

preparation of the DNA for mammalian cell transfections.

Transient expression of IGd H-R I in COS-7 cells and cAMP accumulation

assay. COS-7 cell line was purchased from American Type Culture Collection

(ATCC, Manassas, Virginia). Cultures were transiently transfected with IGpH-R I

construct and with blank pcDNA3.1 vector (negative control) using Lipofectamine

or Lipofectamine 2000 methods (Invitrogen) in 24-well plates accordingly to

manufacturer instructions. Efficiency of transfection was estimated by control

transfections with pcDNA3.1A/5-His-LacZ vector followed by Beta-Gal staining

(Invitrogen). The putative lamprey native ligand for this receptor was not

available at the time of these experiments so human chorionic gonadotropin

(hCG, Sigma-Aldrich, St.Louis MO) was used instead for stimulation of

transfected COS-7 cells. Intracellular accumulation of cAMP was estimated after

3 hours incubation with various concentrations of hCG in Krebs-Ringer buffer

containing 0.1% pyrogen free BSA (Calbiochem) and 0.1 mM IBMX (Sigma). The

buffer was aspirated, cells lysed in 0.1 M HCI or in an Amersham proprietary lysis

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reagent and the concentration of cAMP measured by RIA. The 1-125 cAMP tracer

and cAMP antiserum were a generous gift from Dr. William Moyle (University of

Medicine and Dentistry of New Jersey). Results are expressed as fold increase

over the basal level of intracellular cAMP +/- SEM versus the concentration of

hCG in the stimulation medium.

Effect of IGd H-R on basal levels of intracellular cAMP. To estimate the effect

of IGpH-R I on the basal production of cAMP in transiently transfected COS-7

cells a secreted alkaline phosphatase (SEAP) based reporter system was used.

It showed a higher sensitivity compared to the RIA procedure in experiments

where the rat LH-R was stimulated with hCG or ovine LH (data not shown). The

reporter plasmid pCRE-SEAP contains three cAMP Responsive Elements (CRE)

upstream from the gene encoding a modified human placental alkaline

phosphatase which is secreted into the culture medium upon stimulation by

intracellular cAMP. COS-7 cells were co-transfected with various amounts of

pcDNA3.1/IGpH-R I or empty pcDNA3.1 (0.05, 0.1, 0.2, 0.4, 0.6 and 0.8 pg) and

0.8 pg pCRE-SEAP or pTAL-SEAP (negative control) in 24 well plates with 2 pL

Lipofectamine 2000 transfection reagent (Invitrogen) following the manufacturer's

protocol. After 24 hours incubation, the culture medium was aspirated and

replaced with low serum OptiMEM medium and incubated overnight. The next

day the medium was replaced with serum and phenol red free DMEM (HyClone)

containing 0.1% BSA (Calbiochem) with or without 0.1 mM IBMX. A third

treatment consisted in 0.1%BSA, 0.1 mM IBMX and 5 pM forskolin DMEM for

each IGpH-R I vector amount for normalization of the transfection efficiencies.

After 18 hours of incubation, the medium was collected, centrifuged for 10 min at

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 10OOOg at 4 °C then heated at 65 °C for 30 min to eliminate the intrinsic alkaline

phosphatase activity. A 20 pL aliquot of each sample was then mixed with

reaction buffer (50 mM Tris, pH=8.2) containing 28 mM pNPP (para-nitrophenyl

phosphate) alkaline phosphatase chromogenic substrate then incubated at 37

°C. The optical density was read at 405 nm on a microplate autoreader (BioTek)

at approximately 3 hour time intervals. The results reported here are derived from

end-point optical density measurement data obtained after approximately 24

hours incubation. Absorbance in each well was corrected by subtraction of the

pTAL-SEAP corresponding measurement and normalized in respect to the

corresponding response to forskolin treatment. The same procedural outline was

applied for analysis of the effect of different glycoprotein hormone treatments

(hCG, ovine LH and ovine TSH) on activation of cAMP mediated signal

transduction by IGpH-R I.

Software applications. Assembling of sequencing readings was done with the

pregap4 and gap4 programs from the Staden Package [101]. Sequence

alignments were done using the Clustalw [102] method. The signal peptide

cleavage site was predicted using the SignalP [103] program

(http://www.cbs.dtu.dk/services/SianalP/). Transmembrane regions were

characterized from the thermodynamic profile of the region with programs from

EMBOSS package ( octanol ), the GPCR specific Prosite [104] signature was also

identified using the Prosite motif finder from EMBOSS package (patmatmotifs)

with a locally installed copy of the Prosite database.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5 atgggttgggagcaccgtaggacgtcgtggggggccctggtcgcgctctggctccaggttctggcgct«ccgccgaagg-t.ccaggggggc 9cgtgctccc 104 1 MQWgHBRTBWQALVALWLQVLALPPKVOQ 0 A C 8 P 34

10$ ccgggtgcgtgtgcgcgcacgactggatg»*cttg^tgcagt.gcgtgggcgggAagftcgcl;ggagctg»gcrctgcrtgccggcecacacgcafcacctggi; 204 35 GCVCAHDWKlVLVQCVGGltTI*S:t.8 X.LFABTQ8 LV 6?

205 gttgatgaacacagtt.atcgaaegtgtcccgtcaggagcettctecAggctgag* aacgtcacgRaaategaeatcttagtttceaggeacctgcgeagc 304 68 L M R * V X ERVPSGAPBRLR IIVI® k (d ) iL V S R H L R B 100 ii) 305 ttggaggccatgtccttctcccgtctctcgcagctgcaggacatagaaatcogcaacgccaatcaccttgcctacatccacagggacgcgttccacgagc 404 101 L 8 AM 8 P 8 RL 8 QI 1 QDXE X R X A 8 RLAY IBRDAFHSL 134

405 tgcccagcctcaggttcctgggaatcgccaacacggccattgagtgtttcccgcacgtgggcaagatccagtccacagttctcagcttcatcctcgaaat 504 135 P8LRFX.SXAII7AX8CFPBVGRXQSTVLSFXX.8X167

505 cctggacaaccccatgattgacctcatccccgcaaacgctctgcaaggtctcagctccgggtcgctcatcgtgaggctettcaacaacaagtttgcgaaa €04 168 LDRPMXDLXPAHALQ0l*8SG6LXV------nil LPMVKPAX S-Ki) 200 605 atcgaagacttegccttc aacgcgaccat; stggacctgctggacctccacaacaatgacagactgtgggccatcggcgaggatgccttcagtggaatcc 704 201 Z C B F A F RATH :DLLDLH»MDRLWAlO10AF80IQ234

705 aggagaactctttcatacrtggacgtgtcccgtaoctccgtgcgctcgcttccgcgccacgggctgcageacgtgtggaagctgatggcgaacgccgcgt.g 804 235 EW8riLDVSRTSVRSLPRHOLQHV>fKLMAMAAir267

80 5 gcatctcaagcagctgccccccatgggcagctt-ctcctcgctgtgctccgcqaacctcacctac ;caagccactgctgcgttttctggcaccgcaaggcg 904 268 HLKQLPPHGSrSSLC8 A p L T T >8 8 CCVFlfBRXA 300

905 gcacaggggtgccacgcagacatctacatggaccaggagctccagaacgagagcttcgcctcgagcgocttcgflcttggtgcagtgcgctccggagcccg 1004 301ACOClADIIMDO«LQBKSFAS80rDLVQCAPIPD334

1005 acgccttcaacccctgcgaggacctaatgggccaccagct^;tgogcc^agogtgt^9^:t0^9RSc9^DO^cff

1105 cctcgtoatcctgagcagccgctacaagctggccgtgccacgoCSctcatgtgoaacctggccttcgocgauDotcfcgcatgggogtgtatctactcaca 1204 368 L V IX S 6 RTKLAVPRF A J t C ••<*' I < A FAi>ACH GV*t,I.T 400 T M 3 ...... 1205 atcgcgtccttcgacgtgtacacccgcggcgagtatoacaaceacgccatcgaetggcagacgggcct.cggctgce 9 aetogegggettecteacggt^t 1304 4011 A 8 t 0 VYTRGEYBBBAZOKQFGLGC R X A O P 1 .T V F 434

1305 tcteebcegagetgtcggtottaaegeteaeegtgateaeggtggagegofeggeaeaeeateatctacaccatgcgcctcgaefi 435 88SL8VF'SIrXV2«:VS 3 4KWflT Z Z I I M R L D T M 4 ...... 1405 gcaggoggteacaateabggfigtegocotgggotOfcggegetggtactiggctgccetgeocOtogcfcggc&tcagoagetactccaAggtgagcatttgc 1504 468 Q A V f Z H A S P B * :.fc A 1* V * A A f c * I A , 0 X 8 8 * 8 K V 8 X C 500 T M 5 1505 ctgcccatggacatcgagacgctgccatcgcaggostaegtecaatitcBtectgggcetoaaeatcptggcgttoetegtcfttotgogtgtgctacgogc 1604 501LPMDlETX

1605 acatctactcaacggttaggaacccgagctataacttgggcagccacgatgccaagatcgccaagcgcatggccgtcctcatcttcaccgacttoaectg 1704 535 Xt8fVft8F8Xl(X.eSXI»AXXAKftKA:VXirCX>F1!CS67 1705 catggcgcccatctccttcttogocatectggcggcggoaaagctgccgctcatcacggtgtcgcacaccaagaKjetgctagtgctottetacocgcto 1804 568 MAPI 8 rFAZLA A A KL P L I T W8 HTKILLVLFZPL 600

1805 aa-tgggtgcgccaagccrcttactctaegccatcrfctcRQCaagagcttccgccgagacgtctttatcctgctgagacgcgtggggttgtgcgagcgccgcg 1 9 0 4 601 HACAM PLltXAZFT KSP R R D V P I 1* I. 8 R V 6 I. C 8 R R A 634

1905 cgcagcgccaccgtggccaggtggtgtcaggcggceaccocggeetgcac aatggcagcgtc rcggtcatcatcaggggcggcggtggtggaagccegga 2004 635 QRBRG gVVSGORPai.H P 0 8 V f VI XROGGGG8 QD 667

2005 tctgaatggcggtggtggtgccagcggtggcggeggcggtggtggtggcggtgatggtggttgtggtggcagtggtggtgggottggcgtgaagatggca 2104 6 6 8 LRGGGGA8 GGGCOOCCODOGCGO8 GCGLOVKKA 700

2105 ggcgttggtgccgtggccagcggttggctccgggccccacgcgggctgtcatggtcgtaa 2164 701GVGAVA8Gfri,RAFRGA8W8« 719

Figure 7. The nucleotide sequence and virtual translation of IGpH*R I CDS. The signal peptide is underlined. Transmembrane regions are shaded and labeled accordingly at their N-terminal border; putative glycosylation motifs are marked by rounded corner boxes, the protein kinase putative phosphorylation sites are marked by shaded ellipses. Specific residues and motifs mentioned in text are encircled and labeled.

For identification of protein motifs and patterns the Prosite and InterPro [105]

search services were used with the IGpH-R I amino acid sequence as query. The

maximum parsimony algorithm as implemented in the protpars application of the

PHYLIP package [106] version 3.6 was applied to 1000 bootstrap replica

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (seqboot, PHYLIP package) of the initial amino acid sequence, then the

consensus tree was derived using the consense program from the same

package.

Analysis of the RT-PCR gel images was done with ImageJ application from

NIH [107]. Functional data analysis and graphing were done in Prism (GraphPad,

San Diego CA).

Results

Five distinct primer pairs: GHRS1_128f/GHRS7_512r, GHRS2_256f

/GHRS8_512r, GHRS2_256f/GHRS7_512r, GHRS2_256f/GHRS3_144r,

GHRS7_512f/GHRS8_512r (see Table 5) amplified DNA fragments with sizes

varying between 150 and 800 bp highly similar with Gnathostome GpH-Rs. The

sequencing readings were assembled in gap4 (Staden package) and the

resulting contig approximately 800 bp has been named lamprey glycoprotein

hormone receptor I (IGpH-R I). A smaller number of readings were assembled

into a second, distinct contig which we consider a fragment of a second

glycoprotein hormone receptor (IGpH-R II).

Combination of 3' and 5' RACE reactions resulted in a set of overlapping DNA

clones. The assembled contig was 2705 bp in length. This included a 504 bp 5'

untranslated region featuring two possible translation start sites, an open reading

frame of 2157 bp and a 3' untranslated fragment of 44 bp containing two putative

polyadenylation signals. The most likely translation start site was assigned based

on comparison of the virtual translation of the full transcript with the protein

sequences of GpH-R homologs. Total length of the predicted protein is 719

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. amino-acid residues. The first 29 C-terminal amino-acid residues correspond to

the putative signal peptide predicted by the SignalP program and confirmed by

comparison with other homologous receptors. The extracellular domain including

the signal peptide is 350 amino-acid residues. A 264 residue transmembrane

domain is followed by a putative cytoplasmic tail of 105 residues.

Structural domains of the IGpH-R I were identified based on detection of

specific patterns and motifs and their limits were established relative to the

alignment of the protein sequence with other vertebrate GpH-Rs (Figure 7).

IGpH-R I shows the typical structural domain composition of the Gnathostome

GTH- and TSH-Rs: extracellular domain, rhodopsin-like transmembrane domain

and cytoplasmic domain. In the extracellular domain of the IGpH-R I were also

identified a Cysteine Rich N-terminal domain followed by a Leucine Rich Repeat

(LRR) containing region and a C-terminal Cys Rich Region.

Putative N-glycosylation sites in positions 86, 207, and 285 in the extracellular

domain and one putative N-glycosylation site has been predicted at position 651

in the cytoplasmic of the receptor. Seven hydrophobic helical segments (Figure

7, TM1 to TM7) circa 20 residues in length are linked by 3 intracellular (IL) and 3

extracellular (EL) loops. A disulfide bridge between Cys residues located in EL1

and EL2 is also important for stabilization of the receptor conformation. The C-

terminal end of the 3rd transmembrane helix harbors a protein motif shared by all

members of the GPCR superfamily. The cysteine residue in position 630 matches

the well conserved Cys residue shown to be palmitoylated and anchored to the

cell membrane in the rest of the members of the GpH-R family.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2

0. 8 -

D < 0.4-

0.2 -

brain heart intestine kidney liver muscle testes thyroid

Figure 8. IGpH-R I tissue expression pattern by RT-PCR. Total RNA samples were DNAse treated then their concentration normalized relative to either 28S ribosomal RNA or total RNA amount. RNA samples were amplified in one-tube RT-PCR experiments in parallel with no-reverse transcriptase controls. The graph shows results from at least three experiments with total RNA extracted from different animals. The Y-axis is scaled in arbitrary units representing the ratio of each tissue RT-PCR signal to the RT-PCR signal of testis sample obtained in the same experimental run.

Globally the IGpH-R I has a high sequence identity with Gnathostome

receptors as calculated based on the Clustalw alignment of 46 GpH-R amino-

acid sequence with the virtual translation of IGpHR I coding sequence. The

lowest identity was found for salmon TSH-R (as low as 7% identity) compared to

the highest calculated for canine TSH-R (50% identity). With the exception of

salmon TSH-R the identity varies between 44% (salmon LH-R) and 50% (dog

TSH-R) showing the higher values for TSH- and FSH-Rs. Comparison of IGpH-R

I with fish receptors resulted in the lowest identity values for all GpH-Rs.

Forty nine vertebrate GpH-R protein sequences were used for reconstruction

of the molecular phylogenetic relationships within this class of proteins, using the

GpH-R isolated from sea anemone (Anthopleura elegantissima) [12] as an

outgroup. The resulting tree has the usual topology of the phylogenetic trees

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. most often reported for this family of proteins, with two major clades mirroring the

functional distinction between the TSH-Rs and GTH-Rs. IGpH-R I and the GTH-

Rs form a monophyletic group, the lamprey receptor being the earliest diverged

member of this clade.

The highest level of the receptor transcript was identified in the lamprey testis

tissue using RT-PCR. Detectable levels of the receptor transcript were also

shown in brain, heart, intestine, kidney, liver, muscle and thyroid as depicted in

Figure 8. This figure shows the relative levels of the IGpH-R I signal in different

tissues calculated as percent of the RT-PCR signal found in the testis samples.

A B

■ ■ IGpHR-l ® i.e- □ pcDNA3.1 1

f IS 1« £o 1

DMEM IBMXO.ImM hCG2IU/mL oLH2pg/mL oTSH2(ig/mL

0.010 0.100 1.000 10.000 T re a tm e n t hCG (lU/mL) Figure 9. IGpH-R I cAMP functional assay. (A) cAMP accumulation in response to hCG stimulation of COS-7 cells transiently transfected with a pcDNA3.1/V5/His expression construct of IGpH-R I. On Y-axis the fold increase in intraceilular concentration of cAMP relative to the basal level. On X-axis the concentration of hormone in the stimulation medium (lU/mL). (B) Comparison of activation of SEAP reporter gene activation following the treatment of COS-7 transiently transfected cells with IBMX, hCG, oLH and oTSH. The values on the Y-axis represent the ratio (fold increase) between the results obtained for IGpH-R I and pcDNA3.1 transfected cells, after normalization in respect to the response to the treatment with 5 pM forskolin.

Incubation of COS-7 cells with increasing amounts of human chorionic

gonadotropin (hCG) induced an increase in levels of intracellular cAMP amounts

determined by RIA. However the differences over the basal level was slight

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 9A). Indirect estimation of cAMP synthesis induced by increasing amounts

of IGpH-R I transfected into COS-7 cells showed an increase in the basal level of

cAMP with the amount of IGpH-R I plasmid (Table 2) suggesting that the lamprey

receptor may constitutively induce activation of the cAMP signaling at least in a

mammalian cell line.

Table 2. Constitutive activity of IGpH-R I.

Plasmid amount (fig)

0.05 1.0 2.0 1.0 6.0 8.0

IGpH-R I basal cAMP-b

No IBMX 0.13 0.11 0.16 0.2 0.24 0 .2 7

IBMX 0.2 mM 0 .2 2 0 .1 9 0.23 0.26 0.29 0.32

pcDNA3.1 basal cAMP

No IBMX 0.13 0.12 0.13 0.13 0.14 0.14

IBMX 0.2 mM 0.26 0.23 0.22 0.20 0.22 0.23

Comparison between the abilities of different glycoprotein hormones to induce

the cAMP dependent secretion of the reporter protein in the medium showed a

higher response for mammalian (ovine) LH and TSH compared to chorionic

gonadotropin.

Discussion

The deduced protein sequence of a 719 amino-acid GpH-R (IGpH-R I) was

determined from a 2705 bp cDNA clone isolated from the testes tissue of one of

the most ancient vertebrate species, the sea lamprey. The key motifs, sequence

comparisons and characteristics of the identified GpH-Rs reveals a mosaic of

features common to all other classes of GpH-Rs in vertebrates. The lamprey

GpH-R I was shown to activate cAMP in response to treatment with mammalian

glycoprotein hormones of transiently transfected COS-7 cells.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ■ TSHR Oncorhynchus mykise ■ TSHR Oncorhynchus rhodurus ■ TSHR Morone saxatllis ■ TSHR Oreochromis niloticus ——— TSHR Macaca mulatta ■ .i TSHR Cercopithecus aethiops . TSHR Homo sapiens . i n — TSHR Ovis aries I TSHR Bos taurus ■ TSHR Falls catus I------TSHR Canis famlliarls TSHR Mus musculus • TSHR Rattus norvegicus FSHR Mus musculus FSHR Rattus norvegicus FSHR Mesocricetus auratus i. FSHR Equus asinus I FSHR Equldae 1— FSHR Eq.caballus , i i ... FSHR Ovis arles * ' FSHR Bos taurus FSHR Sus scrota FSHR Macaca fascicularis FSHR Homo sapiens FSHR Cavia porcetlus FSHR Gallus gallus FSHR Cynops pyrrhogaster FSHR Salmo salar FSHR Oncohynchus mykiss f- C GTHRI Oncorhynchus rhodurus GTHRI Oreochromis niloticus FSHR Danio rerio — — LHR Homo sapiens — —- LHR Callithrix jacchus i 1 1 ■■ LHR Meleagris gallopavo LHR Ovis aries LH/CGR Equus caballus LHR Sus scrota LHR Bos taurus LHR Rattus sp. — LHR Rattus norvegicus LHR Mus muscuius i 1 LHR Oncorhynchus mykiss I------LHR Salmo salar — GTHRII Oncorhynchus rhodurus LHR Danio rerio LHR Clarias gariepinus LHR Ictalurus punctatus GTHRII Oreochromis niloticus GpHRI Petromyzon marinus GpHR Anthopieura elegantissima

Figure 10. IGpH-R I molecular phylogeny. Representation of the molecular phylogenetic relationships between members of the glycoprotein hormone receptor protein family. This is a consensus tree derived from maximum parsimony trees calculated for 1000 bootstrap replica of an original set of 49 protein sequences of vertebrate and invertebrate (sea anemone, Anthopieura elegantissima) GpH-Rs. Branches with bootstrap scores lower than 950 are labeled with the corresponding values.

The highest expression of the receptor transcript was demonstrated in the

lamprey testes using RT-PCR. Expression of the receptor transcript was also

shown in lower amounts in brain, heart, intestine, kidney, liver, muscle and

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. thyroid. The high expression of IGpH-R I in the testis and the high similarity with

Gnathostome GTH-Rs suggest that IGpH-R I functions as a receptor for lamprey

GTH.

The total length of IGpH-R I (719 aa) is intermediary between the lengths of

the GTH- and TSH-Rs from Gnathostomes (695 and 750 in average) ([10], [21]).

The differences are due in most part to the differences in the number of residues

in the ectodomain and cytoplasmic domain compared to the other GpH-Rs.

IGpH-R I has the shortest extracellular domain (estimated at 320 residues

without the signal peptide) compared to around 350 residues in GTH-Rs and

around 400 in TSH-Rs. Its cytoplasmic domain is correspondingly the longest in

the family (estimated at 106 residues, compared to around 80 in TSH-Rs, 70 in

LH-Rs and only 60 in FSH-Rs).

The N-terminal half of IGpH-R I has the lowest identity with the vertebrate

GpH-Rs. Potential functionally important residues at different sites in the

extracellular domain of IGpH-R I are unique to this receptor in the context of the

conservation of the global domain organization. This might be of particular

significance for understanding the functional divergence of this class of

receptors. The region is important for ligand binding and contains the

determinants of the specificity of binding of agonists to the GpH-Rs ([89], [88],

[108], [57]).

The Arg in position 192 in IGpH-R I (Figure 7, (i)) corresponds to a well

conserved Lys residue in TSH-R and LH-R. It has been shown that mutation of

the human TSH-R Lys residue in this position to Arg abolishes the ligand

specificity of the receptor leading to increased sensitivity of the human TSH-R to

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. chorionic gonadotropin (hCG) ([14], [27]). Two other interesting residues are the

Lys and Asp residues in position 89 and 91 respectively of the IGpH-R I, located

downstream of a glycosylation site which IGpH-R I shares with TSH-Rs (Figure 7,

(ii)). They correspond to Arg and Tyr residues in the TSH-R consensus (positions

80 and 82 respectively in rat TSH-R). It has been shown [109] that concomitant

mutations Arg80Lys + Tyr82Glu results in a gain of sensitivity of TSH-R to hCG.

The rat LH-R LeuXxxCysXxxGly motif close to exon 1 to 2 junction shown to

be crucial for LH/hCG hormone binding [110] matches the IGpHR I

ValXxxCysXxxGly residues and is distinct from the TSH-R consensus in the

same position. Ala substitution of any of these residues resulted in no hormone

binding although the receptor was successfully expressed on the cell membrane.

Costagliola etal. [111] described a specific motif in TSH-R downstream of the

C-terminal Cys rich domain. The motif with consensus sequence YDY is the site

for sulfation at one of the tyrosine residues and located upstream at the start of

the transmembrane domain. Its presence has been shown to be a requirement

for binding and activation of TSH-R and is suggested to play a similar role in

GTH-Rs. The motif is very well conserved in TSH-Rs, common in mammalian

LH-Rs and present only sporadically in FSH-Rs (where the consensus FDY can

be found instead). This motif is missing in IGpH-R I which is another element

differentiating the IGpH-R I from vertebrate TSH-Rs in apparent contradiction with

the global similarity results.

The glycosylation of the extracellular domain of GpH-Rs has been shown to

be important for the function of these receptors [14]. The presence of only 3

glycosylation sites in the extracellular domain differentiates the lamprey receptor

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. from all other known GpHs which have 6 (TSH-R and LH-R) or 5 (FSH-R) such

motifs in this domain. However, the glycosylation motif in position 86 aligns with a

similar motif in TSH-Rs, the one in position 207 is common to all GpH-Rs while

the NLT glycosylation motif at the N terminal end of the C-terminal Cys Rich

Domain is common in most FSH-Rs.

Tissue expression pattern and functional assay. Initially the receptors for

GpHs seemed to be characterized by their high tissue expression specificity

([14], [89]) in accordance with their tissue specific physiological roles [3]. Later, a

couple of examples of “ectopic” expression of these receptors in mammals have

been described by various authors ([34], [36]). It has become accepted that LH-

Rs are more prone to show extragonadal expression and are less specific while

the FSH-R and TSH-R genes are under a more stringent control over their tissue

expression and binding specificity. Identification and cloning of the GTH- and

TSH-R homologs in fish species seemed to confirm this paradigm, although the

diversity of tissues where the transcripts of GpH-Rs, in particular of fish LH-Rs

could be detected has increased ([55], [10], [38], [112]). The low specificity in

tissue expression of lamprey GpH-R I seems to confirm an evolutionary trend of

decreased stringency of gene expression control in ancient vertebrate lineages.

Nevertheless, its highest level of expression in the testicular tissue of the male

lamprey supports the hypothesis of the involvement of IGpH-RI in the endocrine

control of the reproduction in lamprey in completion of a hypothalamic-pituitary-

gonadal axis as known for all jawed vertebrates ([14], [89]).

Conclusion. Evolution of glycoprotein hormone receptors. Overall, analysis of

the primary structural features of the lamprey GpH-R I reveals characteristics

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. which support the hypothesis of its homology with Gnathostome GTH- and TSH-

Rs. On the other hand it does not offer strong support for its classification in

either of these two groups. The vertebrate GpH-Rs have been divided into two

subclasses based primarily on the functional criterion of implication in two distinct

endocrine control axes in Gnathostomes. It has been speculated that the GpH-R

evolved from possibly ligandless GpH-Rs, whose function was accomplished by

(non hormonal) regulation of their constitutive activity. The increased specificity of

the receptors in later evolved vertebrates seems associated with the decrease in

their constitutive activity in the context of correlated mutations in receptor and

ligand leading to increased thermodynamic stability of the complex [27].

Identification of new orphan receptor members of the GpH-R family (LGR) in rat

and human showing a very low tissue expression specificity and high constitutive

activity seems to confirm this scenario ([3], [113]).

The lamprey GpH-R I described in this paper is the first receptor for

glycoprotein hormones described in Agnathans. It is highly similar and the key

motifs present in its sequence suggest that it is likely a homolog of the vertebrate

gonadotropin and thyrotropin receptors. However, it does not lend itself to a

formal classification in one of these two Gnathostome GpH-R groups. Its

detectable response to a mammalian gonadotropin indicates that it is a functional

receptor that has a role in sea lamprey reproductive physiology. To date there

has been no evidence to support the presence of TSH in this species and only

one GTH-like hormone has been identified [76]. It has been previously shown

that treatment with salmon gonadotropin extract induces an increase not only in

plasma estradiol concentrations but also in plasma thyroxine concentrations in

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. male and female adult sea lampreys [114]. Based on these results it has been

hypothesized [76] that the thyroid and the gonads of the lamprey are under a

pituitary control mediated by the same glycoprotein hormone. From this

perspective the endocrine physiology of lamprey antecedes the evolution of the

pituitary-gonadal and pituitary-thyroid axes in Gnathostomes (jawed vertebrates).

However, further investigations are required for characterization of the ligand

binding and signal transduction activities.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER II

IDENTIFICATION AND CHARACTERIZATION OF THE LAMPREY GLYCOPROTEIN HORMONE RECEPTOR II

Introduction

The search for the origins of the hypothalamic/pituitary/peripheral gland

endocrine systems in the closest relatives of vertebrates, the protochordates

([115], [116], [117], [118], [119], [120]) has provided proofs for the existence of

some of the components of the HPG and/or HPT axes in Amphioxus and/or

Ciona. However, there is limited evidence at this point for their assembling into

functional HPG/HPT axes in these organisms. A brain-Hatschek's pit connection

has been described in amphioxus [121]. However, the hypothalamic-pituitary-

peripheral gland systems and in this context the differentiation between control

mechanisms of thyroid hormone and steroid (gonadal) hormone levels is

considered to be a vertebrate innovation which emerged prior to or during the

differentiation of the ancestral Agnathans into the two main groups of vertebrates

in the actual fauna: the Cyclostomes (lamprey and hagfishes) and

Gnathostomes.

New paleonthological evidence traces the lamprey lineage down to the base

of the phylogenetic tree of vertebrates ([73], [71]). Formerly considered a

'degenerate' branch of the ancestors of vertebrates, the ostrachoderms [71], the

discovery of the fossil remains of an organism surprisingly similar with the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. lamprey but dating 360 mya back in the late Devonian indicates that the

contemporary cyclostomes diverged from the ancient Agnathans much earlier

than usually considered. This discovery places this species in the 'living fossil'

group and cast a new significance over the results obtained from the study of this

organism.

During the last few years, many of the lamprey hormones have been isolated

and characterized ([69], [122]). Studies done on sea lamprey reproduction have

also provided evidence for the similarity between the neuro-endocrine control of

reproductive physiology in this Agnathan and the rest of the vertebrates. Two

forms of gonadotropin hormone releasing hormone (GnRH) have been isolated

from lamprey brain (lamprey GnRH-l [77], and lamprey GnRH-lll [79]). Both

lamprey GnRH decapeptides have been shown by extensive physiological,

anatomical, biochemical and immunological studies ([123], [81], [82], [80])

capable of controlling the reproduction in lamprey and involved also in regulation

of the thyroid hormone levels in plasma. A receptor for lamprey GnRH has been

isolated from the sea lamprey pituitary, its sequence determined and the

functional properties of this protein were recently published [83]. In addition,

numerous efforts have been directed towards isolation and characterization of

the pituitary hormones directly controlling the physiology of the gonads in sea

lamprey: the gonadotropin hormone homologs. These efforts led to the

identification of a pituitary glycoprotein similar in sequence with the beta chain of

the Gnathostome glycoprotein hormones [76]. Analysis of the molecular

phylogenetic data suggests that this molecule is probably an ortholog of the

common molecular ancestor of beta LH, FSH and TSH and diverged from its

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Gnathostome counterpart before differentiation of the different types of beta

GpH-Rs [76]. The functional characteristics of this protein and its role in the

physiology of lamprey in relationship with the well established HPG/T axes

paradigm is currently being investigated.

The molecular cloning attempts to identify the lamprey glycoprotein hormone

receptors resulted in identification, cloning and characterization of a lamprey

member of the GpH-R subfamily (IGpH-R I) [124]. This protein was found

expressed predominantly in the gonadal (testicular) tissue of the mature lamprey.

Its sequence has a number of features which are characteristic of the

gonadotropin receptors. It was shown to have low albeit detectable response to

mammalian gonadotropin hormones but no response to mammalian thyrotropin.

Incidental data obtained during identification of the lamprey GpH-R I suggested

the existence of a second receptor from the same protein family in this animal.

This paper reports the identification in the testes tissue, cloning of the full coding

sequence from the thyroid tissue and characterization of a second sea lamprey

glycoprotein hormone receptor (IGpH-R II, GenBank AY750689) similar to

Gnathostome gonadotropin and thyrotropin receptors.

Materials and Methods

Animals and tissue sampling. The reproductively mature, migrating sea

lampreys were collected during their seasonal migration upstream in the

Cocheco river in Dover, NH. They were transported to the UNH AFAIR

(Anadromous Fish and Aquatic Invertebrate Research) Laboratory where they

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. were kept under a continuous flow of river water at ambient temperature in a

large tank until the time of the experiments.

Animals were killed by decapitation and the tissue samples were immediately

collected, flash-frozen in liquid nitrogen and kept on dry ice until their storage at

-80 °C. The sea lamprey does not have a clearly separated thyroid gland.

Thyroid hormones are secreted from a number of secretory follicles scattered in

the conjunctive tissue located on top of the inferior jugular vein, underneath the

gill basket of the animal [125]. The conjunctive tissue located on the dorsal side

of the jugular vein between gills number 2 and 6 was collected and used for the

preparation of the total RNA used for IGpH-R II cloning and RT-PCR

experiments. The following tissues were also collected and used for

determination of tissue specific expression levels by RT-PCR: testes, liver,

muscle, intestine, kidney, thyroid and brain.

Total RNA extraction and preparation of single strand cDNA. Extraction for

preparation of the total RNA used for cloning the IGpH-R I is described in [124].

Extraction of total RNA for cloning (SO-RACE and full CDS amplification) of

IGpH-R II used lamprey thyroid tissue. The protocol followed the general

guidelines in the Tri-Reagent (MRC) user manual, with a couple of differences.

Briefly, the tissue was removed from the -80 °C freezer and kept on dry ice until

homogenization. Approximately 100 mg of tissue was used for total RNA

extraction. One mL TRI-Reagent was transferred to a glass homogenizer, the

tissue was added to the extraction reagent and immediately homogenized.

Extraction of total RNA for RT-PCR followed the same protocol as used for total

RNA extraction for RACE. The extracted total RNA was run on a non-denaturing

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. agarose gel to asses the quality and relative amount of RNA. The integrated

optical density of the RNA signal on gel was calculated using the ImageJ

scientific image manipulation Java application from NCBI [107], The low

molecular, likely degraded RNA signal on gel was ignored in normalization of the

total RNA amounts used for RT-PCR. The absolute concentration of RNA was

estimated for the sample with the highest quality of RNA as assessed from gel.

RNA sample dilutions were corrected to approximately 200 ng/ pL for all tissues.

Total RNA extracted from testis tissue was used for preparation of the single

strand complementary DNA using a polyA anchored primer containing a Not I

recognition site at the 5' end (Amersham) using the reagents and the protocol

provided by the single strand cDNA synthesis kit from Amersham. Preparation of

ss cDNA for SO-RACE: The single strand cDNA was synthesized using the

Superscript III (Invitrogen) or PowerScript (Clontech) reverse transcriptase.

Multiple samples were prepared at different incubation temperatures, using

Clontech CDSIII universal primer or a reverse gene specific primer for reverse

transcription. cDNA substrate was mixed with the reverse primer and SMART IV

oligo, denatured at 65 °C for 10min then immediately chilled on ice. After addition

of the reaction cocktail containing the reverse transcriptase in the appropriate

buffer, dNTP and DTT accordingly to the manufacturers' instructions, tubes were

incubated at 42 °C or 52 °C for 1 hour then heated 15 min at 70 °C in order to

denature the enzymes. The resulting cDNA product was then treated with 2 III of

RNAse H for 20 min at 37 °C to release the single strand cDNA from the

DNA/RNA heteroduplexes.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PRINTS/GLYCHORMONER fingerprint based degenerate primers and

identification of glycoprotein hormone receptor transcripts. The methodology

used for the design and testing of the degenerate primers for detection of

glycoprotein hormone receptors was described previously (Chapter I, [124]).

Briefly, primers were designed based on the GLYCHORMONER glycoprotein

hormone receptor fingerprint from PRINTS database. Each alignment block of

the fingerprint was translated into a degenerate nucleotide sequence then the

least degenerate 32-mer primer candidates were selected for each element

(degeneracy level ranging between 80 and 2000). The non-degenerate oligos

corresponding to each degenerate primer candidate were generated and a PCR

optimality score was calculated for each of them. A global optimality score for

each degenerate primer candidate was then derived as a weighted average of

the scores of the corresponding non-degenerate oligos. Every 10th non

degenerate oligo was also tested by BLAST local alignment against the GenBank

nucleotide database. The final primers were ranked based on both their predicted

amplification efficiency score and BLAST scores. Calculations were done using

peri scripts coupled with applications from EMBOSS package and the blastcl3

NCBI BLAST client. Around 10 combinations of forward/reverse primers were

used for amplification of lamprey testes cDNA. Resulting PCR clones were

ligated in pGEM-T Easy vector, transformed into JM109 cells, plasmid DNA

extracted with Wizard DNA preparation system (Promega) and sent out for

sequencing.

Screening the sea lamprey whole genome sequencing trace repository. A

lamprey whole genome sequencing project was recently started and the primary

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sequencing data (trace files and base calls) were made available to the scientific

community in TraceDB section of NCBI. The fasta formatted base calls from the

sea lamprey whole genome sequencing project were downloaded from NCBI

trace repository (http://www.ncbi.nlm.nih.aov/Traces/trace.cai) and indexed for

Blast search on the iNquiry bioinformatics server of the University of New

Hampshire. The traces database was then screened for the GpH-R similar

clones using the 'mpiblastn' program with full CDS or fragments of the lamprey

GpH-R I. Blast hits with a similarity score lower than 90% were then used for

screening the public NCBI nucleotide databases using translating blast (tblastx)

for identification of the possible protein similarities.

Amplification of 5' end of IGd H-R II bv Step-Out RACE. The universal primers

5proxB and Udist for amplification of 5' end of IGpH-R II by SO-RACE are

described in [100]. They are complementary with the SMART IV oligo sold by

Clontech so these primers were custom synthesized and used with the SMART

IV from the cDNA library construction kit from Clontech. The Step-Out RACE was

a slightly modified version of the protocol described in [100]. Briefly, the single

stranded cDNA was diluted 1/10 then amplified with the 5proxB primer and the

downstream reverse primer. The product of reaction was checked after 30 cycles

by running a 4 pL aliquot of the reaction on an agarose gel. If only a faint smear

was observed the reaction was stopped there otherwise 5 more cycles were run

to bring the total number of cycles to 35. A 1/20 dilution of these first stage

reactions was used as templates in the second step of the procedure, consisting

of amplification of the DNA product of 1st step with the Udist universal primer and

an upstream (nested) 3' gene specific primer. The number of cycles in the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. second amplification step was 20 with 5 more cycles run if no signal was

observed on the gel (25 cycles maximum). DNA clone samples for sequencing

were submitted at the DNA Sequencing Center of the University of Utah or to

SeqWright (Texas) where they were sequenced using the Big Dye terminator

method. The DNA fragments were preprocessed with pregap4 and then

assembled with the gap4 sequence assembler from the Staden package.

Estimation of tissue expression level of IGpH-R II bv RT-PCR. The one-tube

RT-PCR system AccessQuick RT-PCR system from Promega was used

according to the manufacturers instructions. The tissue specific total RNA

template was normalized in respect to the total amount of RNA as described

previously [124]. The gene specific primers were designed based on the

sequences of the PCR clones corresponding to the transmembrane domains.

Negative control (no reverse transcriptase) RT-PCR reactions were run for each

sample to prevent the artifacts resulting from amplification of genomic DNA.

Detection of Gd H-R in lamprey genomic DNA. A 456 bp DIG labeled DNA

probe was generated by amplification of a fragment of lamprey GpH-R I with

gene specific primers designed against the transmembrane domain of the

receptor (forward primer TCTGCAGGAGGCTCTTCAACAACAAG, reverse primer

TCAGGATGACGAGGATGACGATGG) in the presence of DIG-dUTP:dNTP

nucleotide mix using the DNA labeling PCR kit from Roche, following the

manufacturer's instructions. The labeling efficiency was estimated by comparing

the size of the amplified products with the size of the DNA fragment obtained by

running the same reaction in the absence of modified nucleotides. Lamprey

genomic DNA was extracted from ca 100 mg liver tissue by proteinase K

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. digestion (60 mM Tris, 100 mM EDTA, 0.5% SDS, pH=8.0, 400 pg/mL proteinase

K) followed by two rounds of phenol:chloroform and chloroform extractions,

precipitated with 3 M sodium acetate and 95% ethanol precipitation and

resuspended in TE buffer (pH=7.4). Genomic DNA (40 pg) was then separately

digested with 200 IU EcoR I and Hind III (Promega) respectively in appropriate

buffers at 37 °C overnight and run on a 0.8% agarose gel. The DNA was then

transferred to a Nylon membrane using a non-alkaline procedure and probed

with the DIG labeled DNA probe following the instructions for the Roche DIG-

labeling and detection kit (Roche). The hybridized probes were detected using an

Anti-DIG alkaline phosphatase coupled antibody (Roche) and CSPD

chemiluminescent substrate (Roche).

General sequence manipulation and analysis. Hydropathy score was

calculated and plotted by the 'octanoP program from the EMBOSS package. The

position of the signal peptide was predicted using the SignalP server

(http://www.cbs.dtu.dk/services/SignalP/). The functionally significant patterns

and motifs and the protein domains of IGpH-ll were detected using the InterPro

server (http://www.ebi.ac.uk/interpro/). The intron/exon organization of the

lamprey GpH-R II was determined by running a blastn query against the sea

lamprey trace database with the full or partial coding sequence of the receptor.

The validity of the results were confirmed by analysis of their alignments with the

known exon/exon junctions in the multiple alignment of vertebrate GpH-Rs. The

similarity (percent identity) scores between IGpH-R I, IGpH-R II and the rest of

the vertebrate glycoprotein hormone receptors were calculated using a Python

script.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sequence alignment and molecular phvloaenv. The list of the sequences used

in alignment are in Table 3. Glycoprotein hormone receptor protein sequences

were aligned with muscle v3.6 [126], clustalw v1.83 [102] , t-coffee v5.03 [127]

and probcons v1.1 [128] multiple sequence alignment programs. The

comparative quality scores of the alignments were estimated with the MUMSA

program (v1.0) [129] and used for selection of the final alignment. The highest

relative accuracies of the alignments were found for probcons and t-coffee which

also are recorded with the highest scores in a series of benchmark tests using

standardized input sequence datasets [129]. After visual inspection of the

alignments the probcons output was further used for molecular sequence

analysis due primarily to a more accurate alignment of the N-terminal signal

peptide of the vertebrate GpH-Rs. Regions of the multiple alignment

corresponding to the different functional domains of glycoprotein hormone

receptors were identified and extracted based on the annotations of original

GenBank sequence records and refined by comparison with the results of signal

peptide, protein motifs and hydrophobicity analyses of IGpH-R I and II. Molecular

phylogenies were reconstructed independently for the full amino-acid sequences

cof vertebrate GpH-Rs as well as for the alignments of their functional domains.

First, 1000 bootstrap replica of the initial full length gapped aligned sequence set

were generated (seqboot, PHYLIP package [106] then maximum likelihood

distances calculated (PHYLIP/protdist). The neighbor-joining trees

(PHYLIP/neighbor) were consolidated into one consensus tree

(PHYLIP/consense) and used as the guide tree for maximum likelihood

estimation of branch lengths (PHYLIP/pproml). The same procedure was applied

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. IGpH-R I Deg PCR, PRINTS/'GLYCHORMONER'

j TraceDB

— ► ▼ *•— S I I ■ ■ .TraceDB S3 iiitiiiiiiiimiiiiiiiiimiiiiiiiiimiiiimiiitiiiMiiK * $4 3j s'.. Vfc? • S5

, START SS ST0P

Figure 11. Molecular cloning of lamprey GpH-R II. S1: DNA clone resulted from degenerate PCR amplification (Chapter I); S2: DNA fragment similar with vertebrate GpH-R exon 6 identified in P. marinus WGS trace files; S3 5' step-out RACE product; S4: PCR product obtained with gene specific primers; S5 3' end of IGpH-R II identified in P.marinus WGS trace files; S6: IGpH-R II cds.

to each functional domain alignment (extracellular segment including the signal

peptide, the transmembrane domain and intracellular segment), with the

difference that only 100 bootstrap replica were used for estimation of the

molecular phylogenetic guide tree topology.

Many authors recommend removal of the gap-containing columns and of the

highly divergent positions from the sequence alignments prior to molecular

phylogeny reconstruction. It has been shown that this preliminary treatment of

data may improve the accuracy of the phylogenetic signal, especially for DNA

alignments of distant sequences (see for example [130]). However, the gap-

removal is not recommended in the documentation of the PHYLIP pprotdist

protein distance application [106] so the complete alignments generated by

probcons were used directly, without any extra treatment.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 atgcgggttggaggatcggcgctgacgccgccgctgctgctgctactgcatttcgtggcatgtcactcttggetgtgtccggccaattgccgatgcttag 100 1 MRVCGSALTPPLLLLLBFVACB SWLCPANCRCLE34

101 agcagagggtggagtgcgttgggatgagaaaccttgacataccatcacctctgcccagggacagcaccttcttaaggttcatggacacgatgatagaaga 200 35 QRVBCVGMRNLPIP SPLPRDSTPLRFMDTMIEE67

201 aat'tccggggggtgccttcagtgaccttcccaacattacaaaaatattcttctccatcgccacccagttgcgacacgttcaggcgaatgctttctccaac 300 68IPGGAFSDLPNITKIFFSIATQLRHVQAHAFSN 100

301 ctcgtggagctcaagtacctagaaattaaacacgcaaagaatcttactcgcatacatccgctagctctccaaaacctccccaatctcaagtatctcacca 400 101 LVELKYLEXKHAKNLTRXRPLALQNLPNLKYLTI 134

401 tcaccaacactgggatcagcagcttcccgaacctgcgatgggtccgctccacgatcatgggcttcatectggagatggctgacaatctcaacatgcccet 500 135 TNTGISSFPNLRHVRSTIMGFILEMADNLHHPL167

501 gattcctgccaacgcgtttgaaggcctgagctcagacaccctcatactgaggctgtacaacaacggcatcagagaagtgaagagtcacgcgttcaacggc 600 168IPANAFEGLSS0TLILRLYNNGIREVKSHAFNG 200

601 agccgactccaccgagtggagctgtacagaaatgaacacctgcagaaactcgacaacaacgctttcgccggcgtgctgagaagtccggactacctt^acg 700 201 SRLHRVELYRNEHLQKLPNNAFAGVLRSPDYLDV 234

701 tgtcttggaccgccattgagtccctgccaagccagggcctggacaacctgcgcatcctgcgagccgaagcgaccacgcgcctcaagcatctgccaccaac 800 235 SWTAIESLPSQGLPNLRILRAEATTRLKHLPPT 267

801 ctcggtgctcaaggccctgctcaacacgtegctcacctacccgagccactgctgcgccttccgcggcatgaagcggcagttggggtcgctggagctgcat 900 268 SVLKALLNTSLTYP SBCCAFRGMKRQLGSLELH 300

901 tgcaaccagacgaccctgtaccgctcgcgcatacgcagggcggcggccctgacacagggcaaggcgcctccacbcggcatcgacgccaacggcagcagca 1000 301 CHQTTLYRSRIRRAAALTQCKAPPLGIDANCSSS 334

1001 gcggcgaggcgtggagctctggggaggacgaccggtcggcgcagagcggccactactacttgcccttcctggaggagcccgaggacaccaacgtcggtt't 1100 335 GEAWSSGEDPRSAQSGHYYLPF LEE PEP T N V G F 367

1101 cggggagcagatccgcgtcaccaagagcaagggctcggccaccttcgagcaetcctacggcggtgacccgtttgagttctgcatcccccgagacatcacc 1200 368 GEQIRVTKSKGSATFBHSYGGPPFEFCIPRDIT 400

1201 tgcttgcccaagcccgacgccttaaccccgtgcgaggatatcatgggctacgagtt4:tgcggatgagcatctggttcgtcagcctcctggcgatcgtcg 1300 401 C L P K P P A L TPCEDIKGY E F L r m S IWFVSLLAIVG 434

1301 gcaacctcttcgtcctcctcatcatcctgacgagceactacaagccgacggtgccgcgctttctgatgtgcaacctggccttcgccgacctctgcafcggglia tg g g1400 l 1 435 HLFVLLIILTSHYKPTVPRFLMCNLAFAPLCHGlM G I 4 467

1401 catctacctgctcatcattgccgggttcgacetgtacagccgcaacgagttctacaaccacgccatcgactggcagacggggcccggctgcaacatcgcg 1500 468 IYLLIIAGFPLYSRWEFYMHAIPWQTGPGCNXA 500

1501 ggcttcgtgtcagtgttcgccagcgagctgtcggtctacacgctcatggtgatcacggtggagcgctggcaegccatcaccttcgccatgcgcctcgacc 1600

1601 gcaagatccgcttccgccaggcgctcggcgtcatggccggcggctggctgt-bctcggtggtcctggccggcatgccgctctatggcgtgagcagctactc 1700 535 KIRFRQALGVMAGGWLFSVVLAGMPLYGVSSYS 567

1701 caaggtgagcatctgcctgcoe&tggaqgtcgagacgttegtggegcaggcctaegtggtcaccgtgctcgcgctcaacgtcctggcgttcgtgatcatc 1800 568 600

1801 Itgcgcctgctacgtgcacatttacatcaccgtgcgtaaccccaactacatctcgggcaaccaggacaccaagatcgccaggcgcatggccatactcatctl 1900 601 |cACYVHIYJTVRWPWYZ EGMODTXXARRWAILIF| 634

1901 tcacggacttcatctgcatggcgcccatctcc^bcttcgccatctcggcggcgttcaagctgccgctgatcaccgtcaccaacaccaagatcctggtcgtl 2000 635 TPFICMAPISFFAISAAFKLPLITVTNTKILVV 667

2001 ggtgttcbacccgctgaactcgtgcgcgaatcccttccfcctacgccgtcttcaecaagiiicgttccgccgcgacttcttcatcctcctctgcaagtttagg!gtcttcaccaaginc< 2100 668 VFYPLNSCAHPFLYAVFTKrV F T K r FRRDFFILLCKFR 700

2101 ttctgcgaggcgcgagcgcagctctaccgcgggcagacggcgttgagcgtccacccgtcgggcgtgcgcaacggcagctggacgtcgagccaccgtggca 2200 701 FCEARAQLYRGQTALSVHPSGVRNGSWTSSHRGS 734

2201 gcggtgggacagtgtacacgctcgtcgccctcaacggcagggatggcgcggccggcaagggcggcggcggcggcggcggcgaggtgcccggcgtcgtgtc 2300 735 GGTVYTLVALNGRDGAAGKGGGGGGGEVPGVVS767

2301 gccgtgcgccgcgaatcccggcacatgcgtgcggtgcgccctgbga 2346 768 PCAANPGTCVRCAL* 781

Figure 12. Lamprey GpH-R II nucleotide and protein sequences. Signal peptide is underlined in black. The Signaling Specificity Domain (SSD or ’hinge') is underlined in gray while the transmembrane domain is surrounded by a black rectangle.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Results

Lamprey GpH-R II full length coding sequence. An 879 bp DNA fragment was

cloned and sequenced using degenerate primers based on the PRINTS

glycoprotein hormone receptor (GLYCHORMONER) fingerprint. This fragment

was shown to be highly similar with vertebrate FSH-R, LH-R and TSH-R

receptors and distinct from the previously described lamprey glycoprotein

hormone receptor (IGpH-R I) suggesting that it belongs to a novel, distinct

receptor from the same subfamily. The step-out PCR based RACE experimental

methodology previously used successfully to amplify the 5'end of IGpH-R I

produced truncated fragments. Screening of lamprey genome sequencing base

call database with the fragments of the lamprey GpH-R I resulted in identification

of a sequencing clone containing a short fragment of 78 bp corresponding to the

6th exon of the IGpH-R I but with a significantly different sequence.

The genomic fragment was then used for design of reverse primers

amplification of the 5' end of cDNA as well as forward primers for amplification of

the segment between the exon 6 and the transmembrane domain of the novel

putative GpH-R. Step-out RACE [100] amplified a 583 bp DNA fragment while

the amplification of the exon 6/TMD region resulted in cloning and sequencing of

a 1012 bp DNA fragment. Moreover, screening the lamprey genomic clone

sequences by Blast searches with IGpH-R I intracellular domain resulted in

identification of multiple clones containing the remaining 3'-end fragment of the

IGpH-R II. DNA sequences were then assembled by hand gene specific primers

were designed against the putative translation initiation and stop sites which

were used for amplification of the full DNA fragment corresponding to the IGpH-R

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. II cds. The Kpn I (5' end) and Xho I (3* end) restriction enzyme recognition sites

inserted in the primer sequences allowed cloning of the putative receptor in the

pcDNA3.1/V5-His TOPO (Invitrogen) transfection vector cloning site and

sequencing.

Figure 11. shows a schematic representation of the final full length IGpH-R II

transcript and the methods used for obtaining the different fragments it was

assembled from. The final IGpH-R II coding sequence is 2346 bp long encoding

a 781 aa protein sequence.

Lamprey GpH-R II sequence domains and motifs. The SignalP server

(http://www.cbs.dtu.dk/services/SianalP/. [103]) predicted a most probable signal

peptide cutting site located 23 residues downstream from the translation start

site.

Transmembrane domain was predicted between residues 414 (including

signal peptide) and 681 (Figure 16). Seven putative glycosylation sites (at

residues 78, 114, 199, 275, 302, 330, 724), and 4 protein kinase C (PKC)

phosphorylation sites (at 258, 610, 687 and 730) were identified based on their

specific Prosite signatures [104]. The PRINTS protein fingerprints, GPCR (G-

Protein Coupled Receptors), GLYCHORMONER (glycoprotein hormone

receptors) and TSHRECEPTOR (thyrotropin hormone receptor) were also

detected (Figure 13).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SfOUEMCE: ICoH Oil 3 CRCC* 7D77G4UDirr??UEA t tMC.IH: f l i J a« i& i ^ 1 M trPro im d ip ito B S •> tPfi00027G Fan* PR00237 GPCRRHODOPSN \rr PF00001 TtmJ PS50262 G.PRQTEIN_RECEP_F1 _2 PSQQ237 G_PROTEIN_RECEP_F1J

h iM fin laacha rich repeal , cyrioi«e-rich flanking region, N 'la n M 1PHQ0fQ72- SMQ0GI3 | | LRRNT

bitwrPr* Leoctoe rich repeat IPR001611 Rw«*t PFC0560 | ------— | lRRJ jtflWPK

feiterPr*. Glycopralaki kanaaoa racaplar IPKM2131 : Fan*? PRQ0373 | ------“ ■» » •------1GLYCHORMONER MttrPN

InterPro Thyrotroph* roc optor IPRQC2274 Fan* PR01145 S------m m -m — m — - • ...... m Itshreceptor 1

InterPr • Accessary gone regulator B ipwme 7 4 f

- -

nclPR m M ifn to d unlntegraied G?P§A:l.aMP7giIQ no description G3O8A3.8CUO.I0 no description PTHR23154 LEUCINE-RICH TRANSMEMBRANE PROTEINS rTbft23!Mi'E2Z THYROTROPIN RECEPTOR, TSHR wgnalp signaJ-peptide

3SFS1321 Fan* AG protein-coupled receptor-ike

Figure 13. Protein fingerprints and motifs in the sequence of the lamprey GpH-R II. Output screen from a search session on InterPro database ([131]) using the IGpH-R II as query sequence.

Genomic Southern result and intron/exon composition organization. Depending

on the restriction enzyme used for digestion of the genomic DNA, only one or two

bands were detected by Southern blot using a DIG-labeled probe of ca. 500 bp

corresponding to a fragment of the transmembrane domain of IGpH-R I. Analysis

of the exon number and location in IGpH-R II revealed the typical general

organization of the tetrapod glycoprotein hormone receptor genes: all introns

were found in the putative extracellular domain, the transmembrane and

intracellular domains being encoded by the last exon.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 58 24:25 =25 =25 =26 =23125 =62 =27 381 aa rLH-R L 51 24=25 25 24;26 23 25 ;62 =408 aa rFSH-R f"

56 24=25 =25 =25=26 =23 =26 !63 =471 aa rTSH-R 66 24=25 =25 =25=26=23 =26 162 =418 aa IGpH-R I

58 24i25 =25125=26=23 =26 =62 1488 aa IGpH-R II L

Figure 14. Exon/intron organization of the lamprey GpH-R I and II genes. Position and size of exons are represented in comparison with mammalian (rat) LH, FSH and TSH receptors. The vertical dotted lines mark positions of the introns. The shaded areas indicate the position of the transmembrane domains in the coding sequences of all receptors. The widths of the squares are representative for the relative lengths of each receptor segment but are not drawn rigorously to scale.

Only 9 introns were found, same as in tetrapod FSH-R and TSH-R genes. The

same analysis was done for the IGpH-R I with similar results. The exon/exon

junctions in IGpH-R I were compared with the homologous positions in rat LH-R

and found to correspond down to the last base, with the exception of the

exon9/exon10 junction which in lamprey was found displaced a couple of bases

towards the 5' end of the sequence.

Molecular phytogeny. Figure 17 shows the molecular phylogenetic

relationships derived for the lamprey GpH-Rs with a selection of vertebrate

homologs. The fruit fly (Drosophila melanogaster) GpH-R clearly positions as an

outgroup.

The molecular phylogenetic relationships derived from the analysis of the full

coding sequence of receptors describes the typical grouping of the GpH-Rs in

three main clades: LH-R , FSH-R and TSH-R. The luteinizing hormone receptor

clade is positioned as an outgroup to the monophyletic unit containing the FSH-R

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and TSH-R receptors. Both lamprey receptors form a distinct sister group of the

TSH-R clade.

Table 3. Vertebrate GpH-Rs: database records, domain boundaries and IGpH-RI and II similarities. Total CDS lengths include also the 1st Cys rich box and the signal peptide. The identity scores are calculated relative to the length of the pairwise alignment of the sequences after removal of the gap-only columns.

CDS LRD SSD TMD ID Name Accession Bri GpHRI % aa % % aa % % u % % aa % % ’ number aa GpHRII GpHRI GgHRI GoHRI GpHRI Gp HRI GpHRI GpHRI SoHRII FSHR_Bos_taurus L22319 695 42.7 44.6 ssi 42.0 4 3 .4 8 6 19.8 22.1 2 68 64.9 65.3 70 22.5 31.7 FSHR_BothropsJararaca AY189696 673 42.8 44.9 lit; 40.3 41.6 66 26.4 16.2 268 65.7 69.4 68 21.7 38.6 FSHR_Cavia_porcellus AY082514 695 41.6 44.7 224 42.5 44.7 86 18.7 22.8 268 63.1 64.9 70 21.7 32.7

FSHR_Clarias_gariepinus AJ012647 662 40.0 39.3 2 2 6 35.7 37.7 57 31.7 15.7 268; 63.8 64.9 62 15.8 22.8

FSHR_Cynops_pyrrhogaste AB005587 696 40.9 44.4 224 40.7 41.6 90 18.9 18.6 268 64.9 68.3 68 20.8 33.7 r

FSHR_Danio_rerio AY278107 668 40.3 40.1 •225/; 35.7 37.7 59 29.2 15.4 268 65.7 66.8 62 ; 16.7 25.7

FSHR_Equus_asinus U73659 687 41.5 43.8 224 41.2 43.8 7 8 , . 20.2 22.1 62.3 63.8 22.5 31.7 FSHR_Felis_catus AY521181 695 42.8 44.3 Ills 42.0 4 3 .4 86 18.7 22.1 268 65.7 66.0 22.5 29.7 FSHR_Gallus_gallus D87871 693 41.7 44.6 224 41.2 41.6 8 6 19.8 19.1 268 64.9 68.3 68 20.8 36.6

FSHR_Homo_sapiens A Y 4 2 9 1 0 4 695 41.2 45.0 2 2 4 39.8 44.2 86. 18.7 23.5 268 63.8 65.3 70 21.7 32.7

FSHR_lctalurus_punctatus AF285182 662 40.0 39.3 226 35.7 37.7 57, 3 1 .7 15.7 268 63.8 64.6 62 15.0 22.8

FSHR_Macaca_fascicularis X74454 695 41.4 45.1 224 39.8 43.8 86 18.7 22.8 268 64.2 65.7 70 21.7 34.7

FSHR_Macropus_eugenii AY082002 694 41.3 44.5 224 41.6 41.6 87 18.5 20.4 268 64.2 66.8 68 20.8 37.6

FSHR_Mesocricetus_auratu AY509907 694 40.9 43.3 2 24 4 2.0 43.4 85 18.9 19.9 268 6 2.3 64.6 70 18.3 29.7 s

FSHR_Mus_musculus AF095642 692 41.4 42.3 224 41.6 42.0 85 20.0 19.1 268 6 4.2 63.8 68 16.9 27.7

FSHR_Oncorhynchus_myki AF439405 39.1 38.2 226 36.6 40.4 57 27.0 14.2 269 63.6 61.7 54 12.3 17.8 ss

FSHR_Ovis_aries NM 0010092 695 42.7 44.3 2 2 4 42.9 43.4 86 20.9 22.1 268 64.6 65.3 70 20.8 30.7 89

FSHR_Podarcis_sicula A J 292553 673 38.8 38.0 224 39.4 32.7 67 2 5 .0 16.8 267 59.3 60.8 67 15.8 28.7

FSHR_Rattus_norvegicus NM_199237 692 41.9 43.2 224 41.6 41.6 85 20.0 20.6 268 64.2 64.9 68 20.3 30.7

FSHR_Salmo_salar D Q 8 3 7 298 6 60 39.3 38.5 226 37.0 4 0.8 57 2 7 .0 14.2 268, 64.2 62.7 54 13.2 17.8

FSHR_Sus_scrofa AF025377 695 42.3 43.7 22 4 41.6 43.8 86 18.7 2 0 .6 268 64.9 65.3 70 22.5 28.7

GTHRII_Oncorhynchus_rho A B 0300 05 7 2 4 38.7 36.5 228 38.9 36.1 97 17.3 13.1 268 61.2 63.8 76 18.8 14.5 durus

LHR_Bos_taurus U20504 701 40.4 41.2 225 41.6 41.4 79 23.8 19.7 268 61.9 67.2 77 16.7 20.7

LHR_CallithrixJacchus U80673 676 41.3 41.5 225 40.7 42.3 52 27.3 14.8 268 62.3 68.3 77 18.3 23.4

LHR_Clarias_gariepinus AF324540 710 40.2 37.1 225 42.0 37.9 86 19.8 15.3 268 63.1 63.4 .83 16.5 15.3

LHR_Danio_rerio AY714133 708 39.7 37.4 225 39.8 36.1 93 17.2 13.8 268 64.6 65.3 76 16.8 16.2

LHR_Gallus_gallus NM_204936 728 39.8 40.0 •225, 40.3 42.7 109 15.8 16.0 268 66.4 68.3 81 16.9 19.1

LHR_Homo_sapiens S57793 699 40.2 42.7 225 3 9 .4 44.1 79 2 2 .6 19.7 268 62.3 67.9 77 18.3 21.6

LHR_lctalurus_punctatus AF285181 696 40.1 36.9 225 39.4 37.9 78 2 4 .4 16.4 268 60.8 61.9 71 17.0 16.2

LHR_Mus_musculus NM_013582 700 40.9 41.1 liil 4 1.2 4 1.9 79 2 3 .8 19.7 2 68 62.7 67.5 74 18.3 21.3

LHR_Oncorhynchus_mykiss AF439404 727 38.7 36.5 ills: 39.3 36.1 100 17.0 13.1 268 61.2 63.8 76 18.8 14.5 LHR_Rattus_norvegicus NM_012978 700 40.9 41.3 m. 40.7 4 2.3 79 2 5 .0 2 0 .4 268 62.7 67.9 74 19.2 19.4 LHR_Salmo_salar DQ837299 728 38.9 36.5 228 39.3 36.1 100 17.0 13.1 2 68 61.2 63.8 76 20.5 14.5

LHR_Sus_scrofa NM_214449 696 41.2 42.1 225 41.6 43.2 79 2 5 .0 19.7 268 62.7 67.9 74 16.9 20.0

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3 continued

TSHRB_Oncorhynchus_rho AB030955 793 41.5 40.6 226 44.7 41.0 14 1 . 15.6 21.1 268 65.7 69.0 97 26.5 19.1 durus

TSHR_Bos_taurus N M _ 1 7 4 2 0 6 763 42.0 45.8 226 46.0 43.6 129 14.7 27.5 268 66.4 72.8 87 21.6 23.7

TSHR_Canis_familiaris NM 0010032 764 42.9 45.9 226 46.9 44.9 130 15.4 27.3 268 66.8 72.4 87 24.0 23.7 85

TSH R_Clarias_gariepinus AY129556 777 38.8 39.5 226 43.8 38.3 130 16.2 22.5 268 62.3 65.3 101 17.7 18.9

TSHR_Felis_catus AF218264 763 42.5 46.1 226 46.5 45.8 129 15.5 25.3 268 66.4 7 2 .4 87 23.2 24.6

TSHR_Gallus_gallus AB234613 761 42.2 46.4 226 46.5 43.6 131 18.3 2 7 .8 268 64.6 75.0 85 22.8 22.7

TSHR_Homo_sapiens AY429111 764 42.6 45.9 22 6 46.0 44.9 130 16.2 25.3 268 66.8 73.5 87 22.4 22.9

TSHR_lctalurus_punctatus AY533543 778 38.5 39.5 226 41.6 38.8 131 17.6 23.2 61.9 66.0 101 18.4 17.4

TSHR_Morone_saxatilis AF239761 779 42.6 42.2 226 44.2 44.1 138 15.2 20.9 268 66.4 69.8 93 30.1 20.5

TSHR_Mus_musculus NM_011648 764 42.4 46.4 226 46.5 44.5 130 16.2 28.0 268 66.8 73.1 87 20.8 22.9

TSHR_Oreochromis_niloticu A B 0473 90 778 42.3 42.3 226 43.8 42.3 138 15.9 21.5 268 66.8 70.5 93 27.4 21.2 s

TSHR_Ovis_aries NM 0010094 764 41.8 45.5 22 6 46.0 44.5 130 15.4 25.3 268 65.7 72.0 87 21.6 23.7 10

TSHR_Rattus_norvegicus NM_012888 764 41.9 44.8 226 46.9 43.6 130 16.2 26.7 268 65.3 70.9 87 20.0 21.2

TSHR_Sus_scrofa AF338249 764 42.3 46.2 226 45.1 45.4 130 15.4 26.7 268 66.8 73.1 87 23.2 24.6

GpHRI_Petromyzon_marinu AY750688 719 100 3 9 .9 2 2 6 100 42.3 52 100 12 7 2 6 8 100 6 7 9 111 100 19.9 s T , ... rjSpfeii

GpHRII_Petromyzon_marin A Y 7 5 0 6 8 9 781 39.9 100 226 42.3 100 132 12.7 100 268 67.9 100 101 19.9 100 us

GpHR_Drosophila_melanog U 4 7005 25.5 26.5 223 24.2 26.3 129 9.2 11.4 272 52.2 50.0 76 12.5 15.6 aster

A B Figure 15. Lamprey genomic DNA Southern blot hybridization (A) and IGpH-R II tissue expression levels estimation by RT-PCR (B).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. mouse LH-R hydropathy A less refined phylogenetic

o analysis was included (in the gray

box) to show the relationships o between the glycoprotein

o T hormone receptors (and the new

o CM lamprey members of the

0 200 400 600 Residue subfamily) with a few of the

IGpH-R I (AY750688) hydropathy mammalian LGR receptors. O Reconstruction of molecular O phylogenetic relathionships

O I based on the protein domains

O resulted however, in apparent CMI contradictory results, different 0 200 400 600 Residue

IGpH-R II (AY750689) hydropathy domains showing distinct ptet) o molecular phylogenetic relationships due probably to the o differences in the relative o I functional/evolutionary 0 CM1 constraints acting at the level of

0 200 400 600 Residue different domains.

Figure 16. Hydropathy profiles of lamprey GpH-R I, II compared with mammalian (mouse) LH-R hydropathy. The profiles were generated by the 'octanol' application from EMBOSS package (http://emboss.sourceforge.net/)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. full CDS ' TSHR Icttluna punctata•

LHR Ictalums punctatus —OpHRI Patromyzow i < GTHRII Oncorhynchus rhodurus •OpHRI PMrowyiofi n f LHR Oncorhynchus mykiss LFL LHR Salmo salar — FSHRtetjturapunctBtu* . LHR Clarias gariepinus ■ LUiRlltianocytplcw - LHR Danio rerio -^btRtoMurpiiinGMui — GpHR DrbaopWti maianppaztaf - LHR Homo sapiens 'GpHRCMnortabtWz Ptagaw • LHR Caiiithrix Bacchus • LHR Bos taurus . LHRSusscrofa LHR Rattus norvegicus -LQR4RatMncn«gfaa • LHR Mus musculus -:LGM*om»upt^v • LHR Gallus gallus

. FSHR Claries gariepinus C• FSHRFS " Ictalums punctatus ED • FSHR Dank) rerio -FSHRTatowtt > FSHR Oncorhynchus mykiss • LHRTetrapods ■ c E ri FSHRFS------Salmo salar FSHR Maeaca fascicularis t FSHR Homo sapiens — FSHR Cavia porcelius TSHR Tettapods — FSHR Rattus norvegicus GpHR I PXromyzon marinu* GpHR I Petromyzon marinus __f*— FSHR Mus musculus L— FSHR Mesocricetus auratus GpHR Drosophila mdanogastar j FSHR Bos taurus r f . FSHR Ovis aries L - FSHR Sus scrota TMD I— FSHR Felis catus ■ GpHR I Petromyzon marinus I— FSHR Equus asinus LHRTelrspods — FSHR Macropus eugenii LHRTeteoto _ FSHR Gallus gallus FSHR Bothrops jararaca FSHR Podards siaia ■ FSHRTetrapods - FSHR Cynops pyrrhogaster -TSHR Tate**

■ GpHR I Petromyzon msrinua TSHR tctalurus punctatus — TSHR Clarias gariepinus GpHR OrosopMa melanogasler TSHR Morone saxatiks TSHR Oreochromis nikoticus TSHRB Oncorhynchus rhodurus ID r TSHR Bos taurus GpHR I Petromyzon marinus i. TSHR Ovis aries GpHR tt Petromyzon marinus r TSHR Feks catus FSHR Tetrapods 1* TSHR Canis famiSaris - FSHR Tetoosts _ TSHR Sus scrofa LHRTeteoea TSHR Homo sapiens r - TSHR Rattus norvegicus L. TSHR Mus musculus -TSHR Gallus gallus GpHR Drosophila meianogsiler . GpHRI Petromyzon marinut . GpHRII Petromyzon marinus

GpHR Drosophila melanogaster

— 0.1

Figure 17. Molecular phylogenetic analysis of glycoprotein hormone receptor protein sequences. CDS=full aminoacid sequence, ED=extracellular domain, TMD=transmembrane domain, ID=intracellular domain. Molecular phylogenetic relationship with selected LGR is also represented in the shaded box.

Lamprey GpH-R II tissue expression pattern.The highest response in RT-PCR

experiment was found for the thyroid sample tissue, followed by brain and

gonads. Lower levels of the IGpH-R II transcript were also detected in intestine,

skeletal muscle and intestine and no response was detected for the kidney tissue

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sample (Figure 15 B).

Discussion

A novel glycoprotein hormone receptor (IGpH-R II) was identified and cloned

from the sea lamprey thyroid tissue. The protein sequence as deduced by virtual

translation of the open reading frame is 781 residues in length and highly similar

with Gnathostome GpH-Rs (FSH-R, LH-R, TSH-R).

Its transcripts were detected in thyroid tissue but also in other tissues (gonad,

liver, skeletal muscle, gut and brain). The translated region of IGpH-R II gene

consists of 10 exons alternating with 9 introns. The introns are located

exclusively in the predicted extracellular domain of the mature protein and the

lengths of the exons 2 to 9 are identical with lengths of corresponding exons in

tetrapod GpH-Rs. The molecular phylogeny of the representative members of the

glycoprotein hormone receptor family in vertebrates suggests a close

evolutionary relationship between this receptor and the thyrotropin hormone

receptor subfamily.

This is the second glycoprotein hormone receptor identified in this Agnathan

species. The global identity between the two lamprey GpH-R (40%) was found to

be lower than the identity score calculated between each of these two receptors

and the tetrapod members of this protein family (LH-R, FSH-R and TSH-R). The

lowest identity score was found in respect to the teleost GpH-Rs.

The most obvious feature of the coding sequence of IGpH-R II is the presence

of a long linker fragment (SSD or 'hinge') located in between the Leu Rich

Domain (LRD) of the extracellular segment (ED) and the transmembrane domain

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (TMD). This is one of the longest linker fragment described in all vertebrate

glycoprotein hormone receptors. This comes in opposition with the similar region

of the IGpH-R I which is the shortest SSD/hinge segment amongst all vertebrate

GpH-Rs [124]. The long LRD-TMD linker segment, due to the presence of an

approximately 50 residue insert is a characteristic of the thyrotropin receptors in

vertebrates [27]. This intervening region has been characterized in relation to an

important feature associated with TSH-R that is its cleavage during receptor

maturation and its existence on the cell surface as a functional receptor

composed of two subunits linked by disulfide bridges [132], The receptor

cleavage and reassociation is accompanied by the removal of the 50 residue

insert in the ED/TMD linker region [133]. However, the fine details of the

mechanisms of this process are not yet fully understood, due primarily to the low

similarity between GpH-R paralogs in this region [134]. In fact, the lowest identity

score for lamprey GpH-R II was calculated for the alignments of this region

(Table 3). However, although its length as well as the identity scores bring the

IGpH-R II closer to the tetrapod thyrotropin receptor group, the linker region of

this receptor lacks motifs found to be important for the function of TSH-R in these

animals. For example, the terminal end of the (present) 3rd Cys-rich box upstream

of the first transmembrane segment lacks the Y-[DE]-Y motif related by various

authors to a functionally important Y-sulfation in mammalian TSH-Rs [111].

Lamprey GpH-R I also lacks this motif and in this respect both lamprey

glycoprotein hormone receptors resemble the teleost TSH-Rs which suggests

that functional importance of Y-sulfation is a characteristic acquired later in the

evolution of GpH-Rs, after divergence of Tetrapod lineages.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The presence of 10 exons in the gene structure of the lamprey GpH-R genes

suggests a closer evolutionary relationship of IGpH-R II with thyrotropin and

follitropin receptors in Gnathostomes. Lamprey receptors lack the intra

transmembrane domain introns found in many teleost fish [21] as well as the

extracellular domain 10th intron characteristic to Gnathostome luteinizing

hormone receptor.

The molecular phylogenetic analysis revealed contradictory phylogenetic

signals for functional domains of GpH-Rs. The molecular phylogenetic results are

the result of combined functional and evolutionary constraints acting on

nucleotide/amino acid sequence change during evolution. The interacting protein

domains in protein interacting partners (e.g. GpH/GpH-R ED) are considered

exposed to similar functional constraints and consequently will evolve with similar

rates (see for example [135]), phenomenon usually termed co-evolution of

interacting partners [56]. From this perspective, the extracellular domain reflects

to some extent the evolution of the corresponding glycoprotein hormone. It is

interesting however to notice that the unique lamprey glycoprotein hormone beta

chain identified to date was shown to act as an outgroup to the vertebrate GpH

beta chain clade. One explanation can be related to the finding that correlated

evolutionary change might be due to factors external to the immediate amino

acid composition of interacting partners (receptor/ligand) at the contact points.

Other mechanisms might be the constraints imposed by the intra molecular

interactions between different residues [136], intermolecuiar, functionally

important interactions between the receptors on the cell membrane [137] or the

similarity in expression requirements of the proteins [64].

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Tissue expression pattern of IGpH-R II transcripts shows a wide spread

distribution in several tissues. It is nowadays becoming more and more obvious

the importance of the transcriptional control in the functional evolutionary

divergence of members of a protein interacting network [64]. The tissue

expression pattern of IGpH-R II is in accordance with previous findings about the

specificity of transcriptional control/tissue expression in different taxa indicating a

lower specificity in less evolved organisms ([135], [21], [38]). The high similarity

with thyrotropin receptor is also in agreement with this result, considering that the

thyrotropin receptor has less stringent control of its expression when compared

to gonadotropin receptor counterparts [134].

The genomic DNA Southern hybridization results suggest that IGpH-R I and

IGpH-R II are likely the only representative glycoprotein hormone receptors in

this species. Extensive Blast searches performed within the preliminary lamprey

whole genome sequencing data failed to identify genomic clones belonging to a

different GpH-R (data not shown). We then hypothesize that IGpH-R I and IGpH-

R II are the only members of the glycoprotein hormone receptor subfamily in

lamprey. They are descendants of the thyrotropin receptor-like molecular

ancestors of the GpH-Rs in Gnathostomes and are likely the result of the

genome duplication event hypothesized to have taken place before the

divergence of lamprey lineage ([138], [139], [72]).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER III

FUNCTIONAL DIVERGENCE OF GLYCOPROTEIN HORMONE RECEPTORS.

Introduction

The thyroid and gonadal steroid hormone secretions as well as development

of the thyroid and gonads in vertebrates are controlled by three closely related

glycoprotein hormone (GpH)/glycoprotein hormone receptor (GpH-R) tandems.

Thyrotropin hormone (TSH) is released from anterior pituitary thyrotrophs and

binds to the thyrotropin hormone receptor (TSH-R) in the thyroid modulating the

gene expression in this tissue [11]. Similarly, the pituitary follicle stimulating

hormone (FSH) binds to the cognate receptor (FSH-R) expressed on the

membrane of the testicular Sertoli or ovarian granulosa cells (FSH-R) [15]. The

expression of the receptor for the pituitary luteinizing hormone (LH-R) was

detected predominantly in the Leydig cells of the testes and in theca, interstitial,

granulosa and luteal cells of the ovary [14], Glycoprotein hormones have a

similar molecular organization being dimeric proteins with one subunit (alpha)

present in all GpHs while the beta subunit is specific to each hormone.

Correspondingly, their receptors are highly similar having a large extracellular

domain (ED) containing a Leu Rich Domain (LRD) linked to a rhodopsin-like

transmembrane domain (TMD) through a highly divergent linker region (Signal

Specificity Domain, SSD or 'hinge' region). The third major segment is the

intracellular domain, which also shows a lower similarity score.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The physiology of the hypothalamic-pituitary-thyroid and hypothalamic-

pituitary-gonadal axes has been extensively studied in vertebrates, particularly in

mammals. The hormonal factors of these axes in general and GpH/GpH-R

couples in particular have also been subject to numerous investigations, due to

their involvement in certain pathological conditions in humans [140]. Especially in

the last couple of years, a number of hormones and receptors from this group

have been identified and described in early evolved vertebrate lineages e.g. in

fish (see Chapter I).

However, little is known about the evolutionary origin of this intricate endocrine

control system. It is widely accepted that all glycoprotein hormone as well as their

receptors are descendants of common molecular ancestors and that their

duplication and divergent subfunctionalization started before the divergence of

the vertebrates. The search for the patterns of endocrine control similar with

HPT/G axes in the closest relatives of vertebrates, the Protochordates has

provided important clues but no definite answer to the question of how a high

physiological specificity has emerged from interaction of molecular species which

show otherwise close functional and structural characteristics. Nine forms of

gonadotropin releasing hormone (GnRH) were identified in Urochordates ([141],

[142]). It was also reported that mammalian GnRH is able to induce steroid

synthesis in isolated C.intestinalis gonads [141] and synthetic forms of

urochordate GnRH induced gamete release when injected in adult C.intestinalis

[142]. Although Protochordates lack thyroid follicles, the presence of thyroid like

hormones has been described in blood, endostyle and tunic of the adult

urochordates as well as in their larvae [115]. To date, no evidence has been

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. found for the presence of gonadotropin or thyrotropin like hormones in

urochordates. The closest relative of the glycoprotein hormone receptors

annotated or detectable by Blast searches with GpH-R sequence queries in the

Ciona intestinalis genome (http://genome.jgi-psf.org/Cioin2/Cioin2.hhome.html)

was a homolog of the relaxin receptor LGR7 (data not shown). In

Cephalochordates (amphioxus) GnRH was found [116], LH ([143], [144]) and

TRH immunoreactivity as well as steroid and thyroid hormones (reviewed in

[115]). However, no evidence for the presence of glycoprotein hormone receptors

is available in these organisms. All these data suggest a possible direct

relationship between neuroendocrine factors (GnRH, TRH) and secretion of

thyroid-like or gonadal steroid-like hormones from peripheral glandular tissues

and/or activation of gametogenesis or thyroid like functions in these primitive

organisms [75]. The available evidence offers little support for either the

existence of neural control of a pituitary-like master gland (a projection of the

brain contacting the Hatschek's pit in amphioxus was described as a possible

predecessor of the hypothalamic-pituitary system in vertebrates [121]) or for the

existence of a pituitary/peripheral gland control level.

From this perspective, the appearance of the hypothalamic-pituitary-

thyroid/gonadal axes in vertebrates coincides with the interpolation of a new level

[120] in the respective neural-peripheral gland control hierarchy: the glycoprotein

hormone/glycoprotein hormone receptor, concomitantly with the development of

specialized glandular tissues (the pituitary).

In mammals, the GpH/GpH-R system exhibits two characteristics tightly

related to their proper function under normal physiological condition: the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. specificity of their temporal and tissue expression profiles and selectivity in their

interaction with the ligands ([15], [27], [16], [135]). These characteristics have

evolved during divergent evolution of the ancestral duplicated genes which were

inherently neither specific in their expression nor selective in their ligand

affinities.

The sea lamprey is, together with the hagfish, the only actual representative of

the jawless organisms (Agnatha). It was estimated based on DNA data ([145])

that the Cyclostomes (lamprey and hagfishes) have diverged from the ancestral

Agnathans during the early Paleozoic era, 535-462 mya. Moreover, the fossil

records suggest that the lamprey lineage diverged directly from the ancestral

Agnathans ([73], [71]). This makes this species an excellent object of study for

understanding the endocrinology of the ancestors of present day vertebrates

[72].

Two novel glycoprotein hormone receptors were identified and described in

this species ([124], Chapter III) as well as the putative beta chain of a novel

glycoprotein hormone [76]. This is the only GpH detected so far in lamprey using

molecular biology methods or by screening the lamprey whole genome

sequencing draft data. Molecular phylogenetic relationships between vertebrate

GpH beta chains shows the lamprey sequence removed from the beta TSH, FSH

and LH clade, acting as an outgroup for all vertebrate glycoprotein hormones

beta subunits [76]). The receptor sequences on the other hand are clustered as a

sister group of the thyrotropin receptors when the molecular phylogenetic

relationships are derived using the whole coding sequence data. A different

picture was observed when individual functional domains were used in

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. phylogenetic analysis. The extracellular domain tree exhibits the same topology

as the full coding sequence. The transmembrane domain suggests a phylogeny

where the IGpH-R I acts as an outgroup to vertebrate GpH-Rs while the IGpH-R

II keeps its monophyletic position with the TSH-R sequences ( see Chapter III).

We hypothesize that lamprey GpH-R I and II are homologs of later evolved

vertebrates LH, FSH and TSH receptors, descending from a common ancestor

which was TSH-R-like. The difference in phylogenetic signals for different

domains is due to the functional constraints acting differentially at the level of

different segments of the receptor. The extracellular domain tree topology reflects

a similar pattern of substitution due to the interaction with a single ligand while

the transmembrane domain is more significant in respect to the actual

evolutionary relationships between members of the GpH-R subfamily [130].

Methods and Materials

Glycoprotein hormone receptors. Rat luteinizing hormone receptor was a gift

from Dr. Bill Moyle. Lamprey glycoprotein hormone receptor I was obtained

through molecular cloning from lamprey testes tissue [124].

Hormones. Ovine LH and ovine TSH were obtained from National Hormone &

Peptide Program (NHPP). Human FSH, human CG and the pregnant mare

serum gonadotropin (PMS) were purchased from Sigma (Sigma-Aldrich Corp.,

St-Louis).

Preparation of the chimeric lamorev/rat glycoprotein hormone receptor

chimeric constructs. Rat luteinizing hormone receptor was subcloned in

pcDNA3.1/V5-His TOPO (Invitrogen, Carlsbad CA). The lamprey GpH-R I and rat

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. rat LRD/SSD junction START LrSKBKFTCLX.VfcTLTYPSaCCAr downstream R B I I et«eceteuu9Muatk0ie9«f«cte«t(fteic«tej«tf««etM«c««f««*etfetfcfeetto | | I | | I Plasm id 1 u n iv e rs a l ^ «tcw m u M « ttC T ctH 8 C ttR XICCC (BGH) u p s b v a m mutagenic primers u niversal IMCICC«CCMCTO»CCT»C^> p rim e r C C V r (TV) I I I B I i ( Plasmid 2 STOP lamprey LRD/SSD junction

K p n I A ge I I ctceuifuuiUeaafifectcMCTCCOCClMCtCKCnc

rLH -R IG pH -R I 5 ' p rim e r 3 ' p rim e r

Ctce»>49>A«»»ttO«C9»gcctcTOCTCCOCOACCTC*CCT*CCaUOCCACTOCTOCOmTC

ctcoaakgaaaMttcacsagccCCTaCTCCeCCJUCCTCACCTACCCAAOCCACTOCrOCOrmC

Figure 18. Preparation of chimeric receptors by Splicing by Overlap Extension (SOE-PCR). The figure shows an example of application of the procedure at the level of the rat LRD/lamprey SSD junction.

LH-R DNA fragments were combined in chimeric functional receptors using the

general SOE-PCR (Splicing by Overlap Extension, Figure 18) methodology. The

method or variants of this method have been described by different authors and

applied to a wide range of molecules ([146], [147], [148], [149]).

The two upstream and downstream receptor fragments intended to be spliced

together were first amplified with gene

specific primers and universal primers (T7

and BGH respectively). The 3' reverse primer

for the upstream fragment was usually

chosen as a mutagenic primer, containing at

its 5' end a region complementary to the 5'

end of the downstream fragment. The Figure 19. Agarose gel of the products of SOE-PCR amplification.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. amplifications were done using the Phusion(r) High Fidelity Polymerase from

Finnzymes ((Finnzymes/NEB, Ipswich, MA).

Typical amplification conditions at this step included an initial denaturation at

98 °C for 3 min followed by cycling with denaturation step at 98 °C for 30 sec,

annealing 68 °C 10 sec and extension 30 sec at 72 °C repeated 40 times; the

final extension was carried out for 10 min at 72 °C. The amplification products

were then purified by gel electrophoresis and their approximate amount

estimated by densitometry. The PCR splicing step was conducted in two phases.

In the first phase the receptor fragments were added to the Phusion(r) Master

Mix in amounts adjusted to ensure an approximate 1:1 molar ratio between them

then run through 10 cycles amplification with denaturation step 30 sec at 98 °C,

annealing 10 sec at 60 °C and extension 30 sec at 72 °C. In the second phase,

primers designed against the 5' end of the upstream fragment and 3' end of the

downstream fragment were added to the reaction to a final concentration of 0.2

pM. The same program with annealing temperature increased to 65 °C was then

run for 35 cycles.

The 5' and 3' primers were designed to include a Kpn I restriction enzyme

recognition site and an Age I recognition site site respectively to allow sub

cloning of the full length hybrid receptor. The 5' primers also included the Kozak

consensus for expression in eukaryotic cells. After amplification, the PCR

product was checked on agarose gel. Usually the spliced product was easy to

recognize based on its size compared with the expected total size of the

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. construct. A typical agarose gel of the products of SOE-PCR amplification is

reproduced in Figure 19.

After purification from the gel fragments, the chimeric receptors were digested

with Kpn I/Age I restriction enzymes (5 IU/25 pL reaction, o/n) and ligated into the

Kpn I/Age I linearized pcDNA3.1A/5-His TOPO eukaryotic expression vector and

transfected into the TOP10 (Invitrogen, Carlsbad CA) cells. The plasmids were

prepared for transfection by miniprep (Wizard DNA Preparation System,

Promega Corp., Madison Wl), resuspended in endotoxin free TE buffer and

verified by Kpn I/Age I restriction digest. The amount and quality of plasmid DNA

was estimated by optical density measurements at 260 and 280 nm.

Concentrations of plasmids in the stock solutions used for transfection were

adjusted at the same value based on their OD concentrations and on agarose gel

densitometric measurements of restriction digest products. The pCF3CF2 and

pCF1CF4, containing all the lamprey/rat inter-domain junctions were verified by

sequencing (SeqWright, Texas) in order to validate the experimental

methodology for construction of chimeric receptors, using lamprey GpH-R I gene

specific and universal (T7 and BGH) primers.

The COS-7 cell culture maintenance and transfection procedure. The general

COS-7 culture maintenance was described previously (see Chapter II).

The cAMP dependent signal transduction assay. The experimental protocol for

functional assay of cAMP dependent signal transduction activation by membrane

receptors using the Secreted Alkaline Phosphatase (SEAP) reporter system is

presented in detail in Appendix A. Briefly, the day before transfection the COS-7

cultures grown in T75 flasks in C02 (5%) humidified incubator in DMEM with 10%

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. FBS were cut with 2 mL Trypsin, resuspended in the same medium at 4 x 105

cells/mL and plated on 6 or 3.5 cm Petri dishes or in 12 well plates depending on

the design of the experiment. The next day three hours before transfection the

medium was replaced with fresh DMEM 10% FBS then co-transfected with

receptor construct/ reporter plasmid mixtures at a constant receptor:reporter

mass ratio of 1:7 using the Lipofectamine 2000 transfection reagent following the

manufacturers' (Invitrogen) instructions. The next day the cells were cut with

Trypsin, resuspended at 5 x 105 cells/mL count, plated on 96 well plates at 100

pL/well and returned in the C02 incubator over night. Different layouts for the

placement of the constructs on the plate were used in order to minimize the

systematic errors associated with the position on the plate. In the next step the

culture medium was removed, cells washed with serum free DMEM, overlayed

with 100 ijL serum free DMEM and incubated over night.

Different concentrations of hormones were prepared in a base buffer

containing phenol red free DMEM, 1 mg/mL BSA and 0.2 mM IBMX for all

samples. The serum free DMEM was gently aspirated using a multichannel

pipette and immediately replaced with 100 pL of stimulation medium and

returned in the C02 incubator for overnight. The assay for SEAP activity in the

culture medium was started with centrifugation of the culture plates and transfer

of the medium to new 96 well plates which were incubated subsequently at 65 °C

for 20 min to remove the native alkaline phosphatase activity. The culture

medium was then mixed with 2X SEAP reaction buffer (2M diethanolamine/HCI

DEA, 1 mM MgCI2, pH = 9.8) containing 18 mM paranitrophenylphosphate

(pNPP) chromogenic alkaline phosphatase substrate and immediately transfered

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. to the plate reader (BioTek Inc., Winooski, VT) set at 405 nm for measuring the

initial point in the progress curve.

The optical density measurements were then taken at various time intervals,

increasing from ca. 10 min in the first hour of incubation to 6 h over a period of 24

h. The absorbance data table was immediately saved into a text file and the

automatically inserted timestamps were used to construct the SEAP reaction

progression curves.

Experimental data analysis. The alkaline phosphatase activity (initial rate) is a

measure of the relative impact of different transfected recombinant proteins

and/or treatments on the cAMP dependent signal transduction pathway.

The SEAP reaction progress curve and estimation of initial rate.

o 5 0 0 1000 1 5 0 0 2000 2 5 0 0 3 0 0 0 3 5 0 0

Figure 20. Calculation of the SEAP initial rate. The curve through experimental points was obtained by calculation of the parameters of an empirical saturation function (eq 1) by non-linear regression on all the points of the progression curve. The parameters obtained were used to calculate and plot the linear function f(x)=slope * x (thick line). The line obtained by a linear regression procedure on the first 6 points of the graph is also represented for comparison (thin line).

Initial reactions rates were estimated from the OD vs time progress curves in

two steps. In the first step the parameters of an empirical function describing a

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. saturation kinetics (eq 1, Amax, K empirical parameters, t is time) were estimated

by non-linear regression (Figure 20).

The initial estimates of the parameters were obtained by linear regression on

the first 4 points of the curve (usually corresponding to a reaction time of less

than 2 hours). The initial rates (S) were calculated using the analytical expression

for the 151 derivative of the saturation function using the parameters derived from

regression (eq 2). The initial rate for each experimental sample is the sum of the

contributions of three processes (eq 3): (i) the basal accumulation of cAMP due

to the inhibition of the phosphodiesterase by IBMX (Sbasai), (ii) the accumulation

of cAMP due to the constitutive activity of the receptor construct (S COn stit) and (iii)

the accumulation of cAMP due to the treatment the culture was exposed to

(hormone ligand or forskolin) Streat. The value for SEAP activity determined in the

culture media for concentration of ligand equal to zero (So) represents the sum

between the basal value (S0) and the effect of the constitutive activity of the

receptor constructs ( S COnsm ) (eq 5). The values of S0 were estimated

experimentally as the enzyme activity in the medium samples obtained from

culture transfected with the blank vector (pcDNA3.1 A/5-His TOPO) (eq 6).

f (x) = Amax * ( 1 - exp ( - K * t ) ) (1)

S = f ' (0 ) = Amax * K (2)

( 3 ) (forskolin) (4)

(dose=0) ( 5 )

(pcDNA3.1) ( 6 )

c o n s t i t Sblank ) / ( StotalFsk S q ) ( 7 )

%S - 100 * ( Stotal ~ So ) / ( StotalFsk - So ) (8)

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9/10 10/11 LRD/SSD O Z 3 O C D 0 Cys-ricJ^L^e x o n 9 /1 0 SP LRD SSD TMD ID box 2 ■ / (hinge) SSD (hinge) rat LHR exon 10/11 (rat) Cys-rich 9/10 box 3 SSD/TMD SSD (hinge) SP LRD S S D TM D ID (hinge) GpH-R segment lamprey GpH*R I

PCF36CF23 PCF36CF25 pRF2LP3i o f ; of« j> »o PCF37CF29 PCF37CF31 PCF3CF2 OFHKMBO ■CHKKZD# pCF38CF34 pCF6RF9 OF ofM m m s CIBIMO pRF2LF3_FLAG PCF3RF5 O F I “ ' c m t z j o PCF3CF2 FLAG PCF36CF24 POF36RF13 pRF6LF5 OF*»C=D^ OF-WDO OFWKZDO C PCF8RF9 FLAG PCF37CF30 pCF37RF14 pRF8LF9 OFBBMO OFBKKH* OFBBCQO O L z m z D * pCF3RF5 FLAG PCF38CF33 PGF38RF16 PRF6CF4 OF»»CZK> -OFMCCD* O F w a o COWO

PLF2RF3 pCF40CF22 PCF40CF20 •C 3 D X Z D O OFCZMKZDO o f Ez d * c = * B PLF6CF2 pRF8LF9 FLAG pCF41CF28 PCF41CF26 OFOO>CZ* OFOKZDO O F ! D » C = ) ^ PLF8RF9 PRF6CF4 FLAG pCF42CF28 pCF42CF26 v m m O F O ^ ^ O OFCZKDKZDO o f L d c k i d * PLF6RF5 prLHR FLAG OFCZK0>dDO PCF1LF5 pCF4QCF21. pCF40LF13 i n w i : o f c z w m w o O F D i l M t PCFSLF9 : pCF41CF27 PCF41LF14 •CZKOKIZ* OFLZMMMO O F L Z X liw i pCF1CF4 pCF42CF27 pCF42LF14 ■•CZHMBO. .Of Q 0 * « > O F O » » ^

Figure 21. Diagram of the chimeric rat LH-R/lamprey GpH-R I chimeric receptors.

Differences in means on the dose/response curves were tested using one way

ANOVA (R/oneway. test) and pairwise comparison of means was done using

Welch two sample t-test (R/t.test). All numerical values are reported as

means+/-S.E.M. calculated from experimental outcomes of at least two different

8 0

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. transfections with duplicate treatment samples (dose/response data) or at least

five different transfections in duplicate (constitutive activity data).

Calculations and data plots were done in the R (http://www.r-proiect.org/.

[150]) environment, graphics were subsequently edited in the inkscape SVG

editor (http://www.inkscape.org/) in order to include supplementary information

about the constructs and ligands used. The R scripts written for the data analysis

are available from the author upon request.

Results

The lamprev IGd H-R I / rat LH-R chimeric constructs. Figure 21 shows a

diagramatic representation of the receptor constructs prepared and used in this

study. They are grouped in 6 series, Series A contains domain swapped

constructs downstream of a rat signal peptide; Series B contains the same

constructs with signal peptide replaced by lamprey signal peptide. These first two

chimeric receptor sets lack the FLAG tag and were used for cAMP signal

transduction assays using two concentrations of ovine LH and ovine TSH as

stimuli. The purpose of the experiments involving these constructs was to test for

the possible effect of the signal peptide on the capability of cells transfected with

these constructs to respond to hormone stimulation and to obtain a preliminary

estimate of the effect of gonadotropins versus thyrotropin.

Series C and D were obtained from the constructs of the series A (rat signal

peptide containing chimeras) by insertion of a FLAG tag 'DYKDDDDK'

(GACTACAAGGACGATGACGATAAG) in between the rat signal peptide and the

mature peptide using the SOE-PCR procedure described above. The presence

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of the FLAG tag allows usage of commercial anti-FLAG antibodies for detection

of the protein either on the cell surface by a cell-based ELISA type of assay or

intracellularly by Western blotting. However, the tag was not used in this study at

this stage. Constructs of the series C and D differ by the presence of the lamprey

(series C) or rat (series D) extracellular domain (including SSD) upstream of all

combinations of the transmembrane and intracellular domains.

The constructs of C and D series were used as templates for synthesis of the

chimeric receptors classified under the series E and F which were prepared in

order to test for the effect of the reciprocal exchange of the full or partial signal

specificity domains (SSD) of lamprey GpH-R I and rat LH-R on the ability of the

construct to recognize and respond to luteinizing hormone treatment.

The Signaling Specificity Domain (SSD) or 'hinge' segment is present in all

glycoprotein hormone receptors in between the Leu Rich Repeat Domain (LRD)

and the N-terminal end of the transmembrane domain (TMD).

TMD

ra t LH-R

, . . IVATLTYPSHCCAFRKLPKKE QNFSFSIFENFSKQCESTVRKADNETL i TSAIFEEKELSGHDTTDYGFCSPKTL8CAPEPDAFNPCEDIMGYAFLR

8X0119 axon 10 exon 1 1 ------

lamprey GpH-R I

• CSASLTIPiHCCVmRKAAO (^HADIYMDQELQHESFASSDFDLVCplPEPDAFHPCEDLHGHQLLR

------exon 9 exon 1 0 ------

Figure 22. Details of the rat LH-R/lamprey GpH-R I chimeras SSD level junctions. Arrowed vertical lines mark the splicing sites between rat LH-R and lamprey GpH-R I.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This is a region of poorly defined structural organization considered initially a

mere flexible arm linking the LRD and TMD. However, new experimental

evidence ([23], [17], [151]) suggests that it contains functionally important

residues, critically involved in the selective interaction with the ligand and in

signal transduction. The SSD contains the exon9/exon10 junction in all GpH-Rs,

as well as the exon10/exon11 junction in luteinizing hormone receptors.

■■oLH 1ug/mL EBaoLH 2ug/mL — • BSBoTSH 2ug/mL sX CD i — §O

T> *3 | J lIiu Il HF H i i F a . HriF £t 5 fe S E £ t F 3 & K £ « m 5 <9 a z * £ £ i £ U. u. u. o . s c a a * ft ft ft ft & 4 & * 4 Construct

Figure 23. Effect of oLH and oTSH on chimeric IGpH-R l/rLH-R receptors series A and B.

A detailed diagram of the rat and lamprey SSD is presented in Figure

22.Fragments of the chimeric receptors from series C and D (domain swapped)

were spliced at the level of the LRD/SSD boundary (more precisely 8 residues

upstream the N-terminal end of the SSD), of the exon9/exon10 junctions (rat,

lamprey) and exon10/exon11 (rat) junction. This resulted in 6 different

combinations of the SSD fragments inserted in the original 8 domain swapped

chimeras (Figure 21, E and F).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Preliminary estimation of the Gd H-R chimeras to oLH and oTSH. The effects

of ovine LH and ovine TSH were tested using the constructs of the series A and

B (Figure 23).

pCRE~SEAP+pcDNA3.1 O F flH C M H B t pCRE-SE4P+pCF3GCF23 OFBWKZ* pCR E- SEAP <-pCF36CF24 pCRE-SEAP*pCF36CF25 OFBKKZDO pCRE-SEAP+pCF36RF1 3 OFMOOB* pCRE-SEAPtpCF37CF2S 0 F B K K Z 3 # pCRE-SEAP+pCF37CF3Q OFMC*BO pCRE-SEAP*pCF3?CF31 OFBKKIDO pCRE-SEAP+pCF3?RFl 4 OFM f l pCRE-SEAP+pCF38CF32 OF«XZZ* pCRE-SEAP*pCF38CF33 OFBW) pCRE-SEAP+pCF38CF34 OFMKXZDO pCRE-SEAP*pCF38RF15 OFBWKZ* pCRE-SEAP*pCF3CF2_FLAG oewmmoDO pCRE-SEAP*pCF3RF5_FLAG o f o #c =)♦ pCRE-SEAP+pCF40CFZO H * * OFCimmBO pCRE-SEAP*pCF40CF21 o f o #czx> pCRE-SEAP*pCF40CF2i? OFd l l H pCRE-SEAP+pCF40 LF13 O F C Z K ttZ D # pCRE-SEAP»pCF41CF2G O F C 3 * * * C > pCRE-SEAP+pCF41CF2? O FCZ3#CZX> pCR E-SEAP+pCF41 CFZB QFCZ3C1 — > pCRE-SEAP+pCF41 LF14 OFCZKOKZD^ pCRE-SEAP+pCF42CF2B O F C 3 0 N W O pCRE-SEAPtpCF42CF27 OFE=Kt»C=X> pCRE-SEAP*pCF42CF2B QFM f 0 pCR E-SEAP*pCFBRF3_FLAG O F d M K Z D O pCRE-SEAP+pRFlFLAG pCHE-5EAP*pRF2LF3_FLAG O F[ID C M B K > pCR&SEAF»pRFBCF4_FLAG O F C Z K D ^ ^ pCRE-SEAP+pRF6LF5_FLAG OFOOCZ* pCRE-5EAP+pRFBLF9_FLAG

Figure 24. Analysis of constitutive activity of rat LH-R/lamprey GpH-R I chimeric receptors series C-F. The chimeras of the series C and D (ED, TMD and ID domain swap experiment) are marked by open squares. The units are fold increase over the response to 5 uM forskolin treatment of the same culture, same transfection, expressed as means +/- S.E.M. The asterisks mark the values significantly different from blank (pCRE-SEAP+pcDNA3.1).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The results were calculated as fold increase over the corresponding values

obtained when concentration of stimulus was zero. This implies that the values

obtained here do not account correctly for the relative contribution of the

constitutive activity over the effect of the hormone exposure.

This can be noticed for example in the case of the pRF8LF9 construct (rat LH-

R with lamprey intracellular domain) where the effect of the oLH is

underestimated due to the high constitutive activity of this chimera (see next

section).

However, the results in this experiment indicate a high response for the

constructs containing the rat extracellular domain and suggest that the presence

of the IGpH-R I signal peptide does not impair the activation of the signal

transduction by oLH treatment. The response to the thyrotropin treatment on the

other hand was low for all constructs so this ligand was not used in the next

steps.

Analysis of the constitutive activity. The constructs of the series C to D (FLAG

containing chimeras), D and F (SSD modified chimeras) were used for

assessment of the constitutive activity in COS-7 cells. Figure 24 shows the

SEAP activities in the culture media at concentration of ligand equal to zero,

normalized in respect to the response to forskolin 5 pM treatment in the same

experiment. Three constructs showed a significantly (P<0.05) higher constitutive

activity compared with both constitutive activity of IGpH-R I (pRF2LF3 construct)

and rLH-R (pRF1 construct): pCF40CF20 (rat LH-R with lamprey SSD and ID),

pCF40LF13 (lamprey GpH-R I with rat ED) and pRF8LF9_FLAG (rat LH-R with

lamprey ID).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Response to the treatment with ovine LH. human FSH. human CG and

PMSG.

pRF2LF3_FLAG pCF3CF2_FLAG

o o in in CM CM

8 CM8 CM

o in 8 .ae 8 8

O in

o o

0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 o o v tw L H Dose (ug or IU /mL) ▲ PM8G Dose (ug or IU /mL) □ human CG ■ human FSH pCF3RF5_FLAG O F W t T ~ K> © to CM

8 8 CM CM

O O in in & .a•G 8 8

o ©

0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0

Dose (ug or IU /mL) Dose (ug or IU /mL)

Figure 25. Response of domain-swapped rat LH-R I lamprey GpH-R I chimeras (series C, lamprey ED) to oLH, hFSH, hCG and PMSG.

Figure 25 and Figure 26 show the dose/response curves obtained by

treatment of each of the chimeric constructs from series C and D with four

glycoprotein hormones (ovine luteinizing hormone oLH, human chorionic

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. gonadotropin hCG, human follicle stimulating hormone hFSH and pregnant mare

serum gonadotropin PMSG).

pRF6LF5_.FLAG^ pRF8LF9_FLAG O F C Z K O X Z Z ^

.2 S C ts 8 -

8 - S -

- 1------1------i------1------!------1- ~I------1-----1------1----- 1---T"

0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 o ovkw L H Dose (ug or IU /mL) ▲ PMSO Dose (ug or IU /mL) □ human CO pRF6CF4_FLAG ■ human F8H prLHR_FLAG O F d M M o f c z w k id o O in CM CMs I

o in

ts 8 8

O O m m

o ©

0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0

Dose(ugorlU/mL) Dose (ug orlU/mL)

Figure 26. Response of domain-swapped rat LH-R I lamprey GpH-R I chimeras (series D, rat ED) to oLH, hFSH, hCG and PMSG..

Analysis of Series C (with lamprey extracellular domain) chimeras did not

result in a significant response for any of the hormones tested with one

exception: the response of the pCF3CF2_FLAG construct (lamprey GpH-R I with

rat TMD) to luteinizing hormone oLH stimulation.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Receptors of the series D (rat extracellular domain) resulted in a high signal

detected in response to stimulation with any of oLH, hCG and PMSG hormones.

The follicle stimulating hormone hFSH was inactive with the exception of one

construct, pRF8LF9_FLAG (rat LH-R with lamprey intracellular domain ID) for

which a signal almost as high as for hCG and PMSG was detected.

Effect of SSD modification on the response to ovine LH treatment. 23

lamprey/rat chimeras were prepared by reciprocal exchanges of the fragments of

the SSD segment of both receptors applied to each of the constructs in series C

and D.

p€F36CF23 0 F H K M B 4 pCF36CF24 O F M O «

pCF37CF29 pCF37CF30 O F B W X Z D *

- O - pCF3BCF32 O F I pCF 38CF3aO F eO G Z )^

125 -

O. a

0 0.1 0J «3.3 0,4 0.S 0.« 0.7 0.« 0.9 i 0 0.1 0.7 0.3 0.4 0.5 0-ft 0.7 0.0 0.9 1 Dose (ug/m l ovine LH) Dose (ug/mL ovine LH)

Figure 27. Effect of SSD manipulation on the response of chimeras containing lamprey ED to oLH stimulation. Diagrams of the chimeric receptors are represented above the dose-response plots.

These constructs were then used for screening the change in their response

upon exposure of the transfected cells to mammalian (ovine) LH. Only the most

significant results are reported here. Insertion of the rat SSD in the wild type

IGpH-R I resulted in increased response to hormone treatment (Figure 27 left

panel, pCF36CF23).

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. This increase was lost when the N-terminal fragment of lamprey SSD (see

Figure 22) replaced the corresponding region of the rat SSD (pCF37CF29). The

responsiveness of the construct to oLH was restored after removal of the rat

fragment corresponding to exon 10 (pCF38CF32), this chimera inducing an even

higher stimulation of SEAP secretion than the rat SSD only receptor at higher

concentrations of ligand.

A similar pattern was observed when the same manipulations of the SSD were

applied to the lamprey ED/ratTMD hybrid (Figure 27, right panel): insertion of the

rat SSD upstream the rat TMD (pCF36CF24) resulted in increased response of

the chimera to oLH treatment. Replacement of N-terminal fragment with lamprey

sequence (pCF37CF30) resulted in an unresponsive construct. Further

elimination of the 10th exon sequence of rat (pCF38CF33) restored the activation

of the construct at even higher levels.

Discussion

Construction of chimeric proteins is an experimental tool that is widely used in

the study of functional properties of glycoprotein hormone receptors. This

approach was particularly effective in identification and localization of the

structural determinants of the ligand binding selectivity among thyrotropin,

lutropin and follitropin members of this subfamily of receptors ([89], [135], [14],

[17]). Depending on the specifics of the experimental designs applied, smaller or

larger fragments of one receptor are inserted into a second one and the

properties of the new construct are evaluated by different experimental methods

in respect to the properties of the original proteins as a reference.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Application of this methodology to investigate the functional divergence

between a lamprey GpH-R (IGpH-R I) and a mammalian (rat) GpH-R (rl_H-R)

resulted in identification of specific combination of domains exhibiting functional

properties which are distinct from their parent receptors (lamprey and rat) in

respect to both ligand selectivity and constitutive activity. This suggests that

evolutionary change at the level of individual domains did not altered

fundamentally their original functions, but receptor selectivities have been refined

by subtle correlated changes in multiple domains under the specific constraint of

binding of increasingly divergent ligands.

Although not always explicitely stated, this experimental approach is based on

an assumption of a non-random effect of the transfer of a particular protein

fragment from one molecule to another, i.e. on an assumption of functional

homology between the protein segments under investigation. This relies

ultimately on the concept of the proteins as strings of domains, each domain

being a functional unit, exposed to specific evolutionary constraints.

In the case of the GpH-R subfamily of receptors, the main structural/functional

units usually characterized are the extracellular domain (ED), the transmembrane

domain (TMD) and the intracellular domain (ID). Their functions are, respectively,

the binding of the ligand, the transfer of signal to the intracellular medium and the

regulation of receptor silencing and internalization.

Experimental evidence collected especially in the last decade suggested a

finer functional specialization within these broader structural units, leading to

identification of the roles of conserved residues within the Leu Rich Repeat

Domain (LRD) [152] and the Cys Rich boxes 1,2 and 3 [153].

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Moreover, the formerly neglected poorly conserved, low complexity or

intrinsically disordered protein regions have received new attention after the

collection of experimental evidence indicating that these regions may have an

important role in the protein function both in general [154] or in the particular

case of the LRD/TMD linker of GpH-Rs ([155], [23]).

Lamprey GpH-R is the most distant member of the vertebrate group of

glycoprotein hormone receptors identified to date. A glycoprotein hormone beta

chain was also described in lamprey and its properties are being investigated but

no data is available at this point on the binding to IGpH-R I or signal transduction

activation [76]. The IGpH-R I domains were assigned based on a multiple

alignment with the vertebrate FSH-Rs, LH-Rs and TSH-Rs but the only support

for their putative roles is the similarity scores with homologous regions of

vertebrate GpH-Rs [124]. The identity scores between lamprey and rat domains

are as follows (see Chapter III): 40% for LRD (comparable with the identity

scores calculated between members of the vertebrate GpH-Rs which varies

between 39% and 46% [135]), 25% for SSD (low identity score but one of the

highest found between IGpH-R I and all other vertebrate GpH-Rs), 67% for TMD

(again, comparable with vertebrate scores 68-72%), and 20% for ID (the most

divergent segment).

Given the lack of mechanistic data for IGpH-R activity and the large

evolutionary distance separating them, it is difficult to draw an indisputable

parallel between the functions of the similar sequence segments of these two

receptors. Moreover, the possibility exists for the chimeric receptors to exhibit

new emergent properties, not directly related to the properties of the parent

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. receptors, properties resulting from novel chains of interactions between residues

and domains put in close proximity for the first time [156].

Taking into account these factors, we attempt to interpret the effects of

glycoprotein hormones on the chimeric lamprey/rat receptors in three respects:

(i) as indicative of the functional roles of the lamprey GpH-R I segments formally

identified based on their similarity scores in the MSA, (ii) as providing further

mechanistic information in respect to the roles of the rat LH-R domains and (iii)

as emergent properties, uniquely characteristic to the respective lamprey/rat

combination of domains.

The traditional concept on the mechanism of ligand binding and signal

transduction (MODEL A) assigns distinct roles to the extracellular and

transmembrane domains of the GpH-Rs. The ligand contacts both the

extracellular domain at the level of the Leu rich repeat (LRD) and the

transmembrane domain (TMD) at the level of extracellular loops 2 and 3 . The

specificity of interaction is encoded in a small number of residues located on the

internal face of the horse shoe shaped LRD (reviewed in [14], [157]).

Identification of naturally occurring mutations in the transmembrane domain of

human FSH-R resulting in an increased sensitivity of the receptor to hCG

challenged the common understanding of the role of the serpentine region in the

mechanisms determining the specificity of interaction of glycoprotein hormone

receptors with the cognate ligands. The signal transduction activity of the

receptor (TSH-R then extended to all GpH-Rs) is inhibited by the extracellular

domain in the absence of the ligand (MODEL B [158]). The specific binding of the

ligand induces a conformational change in the LRD which in turn results in

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activation of the signal transduction via interaction with the extracellular loops of

the TMD. The specificity in this model is also encoded in the extracellular

domain. However, this model does not require direct interaction between the

ligand and the transmembrane domain [135].

A third model of the specificity of activation of GpH-Rs by their ligands

(MODEL C, [155], [23], [17]) also indicates the Leu rich repeat domain as the

primary site for hormone binding but instead of restraining the specificity

determinants to the LRD (as in MODEL A) or delegating the specificity of

activation to a 'locked' state of the transmembrane domain (as in MODEL B) it

describes the LRD, 'hinge' and TMD as an integrated functional unit, the

selectivity of the receptor being the resultant of complex interactions between

these three structural units and the ligand during binding and activation of the

receptor. The ED/TMD linker segment is identified in this case as a Signaling

Specificity Domain (SSD).

Response of chimeric receptors to gonadotropin and thyrotropin. In this

experiment a preliminary cAMP signal transduction assay was performed in order

to screen for gonadotropin/thyrotropin response of domain swapped constructs.

Half of the constructs have the lamprey signal peptide at the N-terminal end.

Constitutive activity profile of chimeric receptors suggests a role of the

LRD+SSD unit in modulating the intrinsic constitutively activated state of the

TMD. The constitutive (basal) activation of cAMP pathways by the ligand-free

receptor is the result of the transmembrane domain being locked in an activated

state in the absence of the ligand. The basal activity of wild type constructs pRF1

(rLH-R) and pRF2LF3 (IGpH-R I) show low levels if any. The results in this

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. experiment can be summarized as follows: (i) lamprey intracellular domain (IID)

induces increased constitutive activity in rLH-R. (ii) the concomitant presence of

the ID and SSD segments of the lamprey in rat receptor seems to act

synergistically to induce a very high level of basal activity, (iii) this effect seems to

be dependent primarily on the presence of the rat extracellular domain.

Analyis of domain-swapped mutant response to gonadotropins indicates a role

of the transmembrane domain in ligand selectivity mechanisms. All chimeras

were modified to include the rat signal peptide at the N-terminal end to ensure

the homogeneity of protein expression on the cell surface. Four gonadotropin

hormones were used in the cAMP assay in order to test for the changes in

selectivity of binding and activation by gonadotropins of domain swapped

receptors. Insertion of the rat transmembrane domain into the wild type lamprey

receptor resulted in increased sensitivity to LH only. Presence of the lamprey

intracellular domain downstream the rat LRD+SSD+TMD combination altered the

selectivity of the construct in respect to follitropin.

The rat SSD is capable of transferring the sensitivity to lutropin in the lamprey

receptor. The presence of the rat exon 10 inhibits the activating effect of the

lamprey N-terminal SSD + rat C-terminal SSD. In the last series of experiments,

the effect of different reciprocal exchanges at the level SSD ('hinge') fragments of

IGpHR-l and rLH-R on the response to mammalian lutropin was measured in an

attempt to determine the possible role of this region in receptor IGpH-R I

selectivity. Exon 10 is dispensable for the binding of hCG by hLH-R [89].

In summary, the results indicate that although the lamprey GpH-R I shows a

low capability to be activated by a tetrapod gonadotropin (ovine LH), replacement

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of the C-terminal half of its Signaling Specificity Domain with its rat counterpart

drastically changes its responsiveness to oLH treatment. This indicate that the

functionality of the glycoprotein hormone receptors was remarkably well

conserved in spite of the hundreds of millions of years of divergent evolution.

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. SUMMARY AND CONCLUSIONS

1. Two lamprey glycoprotein hormone receptors (IGpH-R I and IGpH-R II)

were identified and cloned. They are the earliest diverged glycoprotein

hormone receptors identified to date in vertebrates.

2. Analysis of IGpH-R I and II sequence and phylogenetic relationships

with the vertebrate homologs suggests that their common ancestor was a

TSH-R like molecule. The presence of a single ligand in lamprey

constrained evolution of their extracellular (binding) domain to a similar

pattern of substitutions. Still, features which are characteristic to individual

classes of receptors in more evolved vertebrates can be recognized in

lamprey which suggests that they have evolved initially in the absence of

specific ligands.

3. Functional roles of the lamprey GpH-R I domains are conserved

compared with its Gnathostome homologs. The ability of different

glycoprotein hormones to activate chimeric lamprey/rat receptors suggests

that the selectivity of the GpH-Rs in respect to their ligands is not

controlled exclusively by a single domain but is the result of specific

interactions between domains. These interactions were refined during

millions of years of correlted evolution of the receptors with their cognte

ligands under particular intramolecular, intermolecular and physiological

constraints.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDICES

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX A. IACUC Approval Note

U n iver sity o/N ew H ampshire

July 27, 2005

Sower, Stacia Biochemistry Molecular Biology Rudman Hall Durham, NH 03824

' IACUC # : 040701 Original Approval Date; 07/21/2004 Next Review Date; 07/21/2006 Review Level: C

Project: Neuroendocrine Control of Reproduction in Fish

The Institutional Animal Care and Use Committee (IACUC) has reviewed and approved your request for a time extension for this protocol. Approval Is granted until the "Next Review Date" indicated above. You will be asked to submit a report with regard to the involvement of animals in this study before that date. If your study is still active, you may apply for extension of IACUC approval through this office.

The appropriate use and care of animals in your study is an ongoing process for which you hold primary responsibility. Changes in your protocol must be submitted to the IACUC for review and approval prior to their implementation.

Please Note: 1. All cage, pen, or other animal identification records must include your IACUC # listed above. 2. Use of animals in research and instruction is approved contingent upon participation in the UNH Occupational Health Program for persons handling animals. Participation is mandatory for all principal Investigators and their affiliated personnel, employees of the University and students alike. A Medical History Questionnaire accompanies this approval; please copy and distribute to all listed project staff who have not completed this form already. Completed questionnaires should be sent to Dr. Gladi Porsche, UNH Health Services.

If you have any questions, please contact either me at 862-2726 or Julie Simpson at 862-2003.

For the IACUC,

Research Conduct and Compliance Services, Office of Sponsored Research, Service Building, 51 College Road, Durham, NH 03824-3585 * Fax: 603-862-3564

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX B. cAMP Assay Using the pCRE-SEAP Reporter System

Principle of the Assay

Functional assay for the study of a recombinant protein (usually a G-Protein Coupled Receptor) which activity affects the cAMP dependent signal transduction pathway. The recombinant DNA encoding the protein of interest is transiently co transfected in COS-7 cells together with a reporter plasmid containing the gene encoding a modified human placental alkaline phosphatase engineered to be secreted into the culture medium (Secreted Alkaline Phosphatase, SEAP or SeAP). The SEAP gene is under control of a battery of CRE (cAMP Responsive Element) cis-Regulatory Elements. Activation of the SEAP transcription reflects the stimulation of a signal transduction cascade induced by the increase in concentration of intracellular cAMP second messenger via phosphorylation of the CREB (cAMP Responsive Element Binding) regulatory protein. The increase in concentration of cAMP may be the result of up-regulation of its biosynthetic pathway (active) or the result of down-regulation (inhibition) of its degradation pathway (passive). The reporter protein (SEAP) is secreted in the culture medium where its activity can be determined by standard enzyme activity assay procedures. The alkaline phosphatase activity is a measure of the relative impact of different transfected recombinant proteins and/or treatments on the cAMP dependent signal transduction pathway.

Materials

The amounts required are calculated for the transfection of a 10 cm plate of COS-7. The number of cells and correspondingly the number and size of the plates needed depend on the particular design of the experiment (number of transfection constructs, number of treatments, number of experimental replica etc). The volume of reagents and the amounts of DNA are therefore to be adjusted accordingly. Use the surface ratios in Table 4 below or refer to the Lipofectamine2000 reagent documentation for calculation of the actual amounts needed for your experiment. Please note that any experiment should involve the co-transfection of at least two plasmid constructs with the reporter: the recombinant protein of interest and the (blank) transfection vector used for cloning of the experimental construct.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. COS-7 CULTURE * 1 HUIII DMEM 10% FBS, 37 °C, 5% CC>2 incubator

PLATING 5 ca. 4 x 10 cells/mL (ca. 95% confluence), o/n

CO-TRANSFECTION Receptor:pCRE-SEAP reporter plasmid 1:7 (w:w), Lipofectamine 2000

TRANSFER 96 WELL PLATES 5 5x10 cells/mL (ca. 95% confluence) 100uL DMEM 10% FBS, o/n

SERUM STARVATION + + + > + + + + ♦ ♦♦ ♦ ♦♦♦♦ Wash, overlay with 100uL DMEM, no FBS, o/n

STIMULATION various concentrations ligand, 5uM forskolin, 100uL DMEM, Phenol Red free, 1mg/mL BSA, 0.2mM IBMX

Progress curve MEDIUM SAMPLES and initial rate Centrifuge, collect medium new 96 well plate, incubation 20min, 65 cfc

OD SEAP ACTIVITY 50uL 2X DEA buffer, pH=9.8,18mM pNPP + 50uL medium, incubate r.t., kinetic OD measurement @ 405nm TIME

Figure B.28. Outline of the cAMP functional assay using the pCRE reporter system.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4. Transfection reagent amounts for pCRE-SEAP/receptor co-transfection. Amounts depend on the culture vessel used (* for a 1/10 receptor/total DNA amount used in transfection - subject to optimization)

Vessel Surface area approx# of assay Shared reagents DNA transfection (cm2) samples (wells/96well plate) plating dilution medium DNA total DNA r e e f) DNA rep O Upofectamlne2 medium 000

96 well 0.3 1 100 pL 2x25 pL 0.2 pg 20 ng 180 ng 0.5 pL

24 well 2 6 (0 row) 500 pL 2x50 pL 0.8 pg 8 0 ng 720 ng 2.0 pL

12 well 4 12 (1 row) 1 mL 2x100 pL 1.6 pg 160 ng 1440 ng 4.0 pL

6 well 10 24 (2 rows) 2 mL 2x250 pL 4.0 pg 400 ng 3.6 pg 10 pL

3.5 cm plate 10 24 (2 rows) 2 mL 2x250 pL 4.0 pg 400 ng 3.6 pg 10 pL

6 cm plate 20 60 (5 rows) 5 mL 2x500 pL 8.0 pg 8 0 0 ng 7.2 pg 20 pL

10 cm plate 60 2 plates 15 mL 2x1.5 mL 24 pg 2.4 pg 21.6 pg 60 pL

Protocol

Step 1: Plating

• Aspirate the medium from the T75 flask containing the COS-7 stock culture. • Wash the cells with 10 ml_ PBS or DMEM • Cut cells with 2 ml_ Trypsin; incubate ca 20 min at 37 °C in the humidified C02 incubator • Wash the flask with 10-20 mL DMEM 10% FBS and transfer suspension to a sterile 50 mL centrifuge tube • Resuspend the cells by pipetting up and down • Count cells • Dilute with DMEM 10% FBS to a final cell count of 4x105 cells/mL • Transfer 10 mL cell suspension to a 10 cm plate • Mix the cells by gently moving the plate up/down and left/right to ensure an uniform distribution of cells on the surface of the plate Incubate o/n at 37 °C , 5% C02

Step 2: Co-transfection

• 2 h before transfection aspirate the medium and feed the cells with 10 mL fresh DMEM 10% FBS

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. • Prepare the DNA solution: 21.6 pg pCRE-SEAP reporter plasmid and 2.4 pg receptor plasmid (24 pg total amount of DNA 1:9 w/w receptonreporter ratio) in 1 mL OptiMEM. • Mix gently the tube containing the stock Lipofectamine2000 (LFA2000) transfection reagent suspension • Prepare the LFA2000 suspension: 60 pL LFA2000 in 1 mL OptiMEM. Mix gently and incubate 5 min at r.t. • Mix the DNA and LFA2000 in OptiMEM solutions. Mix gently and incubate 20 min at r.t. • Remove the plate with COS-7 cells from the C02 incubator and carefully add the DNA/LFA2000 transfection complexes suspension to the cells • Mix the cells by gently moving the plate up/down and left/right to ensure an uniform distribution of transfection complexes on the surface of the plate. • Return the plate in the C02 incubator

Step 3: Re-plating

• Aspirate the medium+transfection mix • Wash the cells with 10 mL PBS or DMEM • Cut cells with 2 mL Trypsin: incubate ca 20 min at 37 °C in the humidified C02 incubator • Wash the flask carefully with 10 mL DMEM 10% FBS and transfer suspension to a sterile 50 mL centrifuge tube • Resuspend the cells by pipetting up and down • Remove an aliquot of the cell suspension (ca 200 pL) and count cells • Dilute with DMEM 10% FBS to a final cell count of 5.105 cells/mL • Transfer the well suspended cells to a sterile multichannel tray and immediately start pipetting 100 pL cell suspension to the 96 well plate. Keep cells in suspension by pipetting up and down a few times before each column (row). • The 96 well plate should not need mixing. Incubate the 96 well plates in the C02 incubator o/n

Step 4: Serum starvation

• Gently aspirate the culture medium from the 96 well plate. Use a multichannel pipette with P200 tips. Be careful to touch the cell layer as gently as possible. Proceed with one plate at a time or even better with 1/2 plate at a time. Have a second multichannel pipette and medium tray ready and immediately add the replacement medium to the empty wells. • Do first a wash (PBS or DMEM) then overlay cells with plain DMEM • Return cells to the C02 incubator for o/n incubation

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Step 5: Treatment

• Prepare the base stimulation medium: o Phenol-red free DMEM - 10.8 mL per 96 well plate o 10X BSA (10 mg/mL in PR-free DMEM) - 1.2 mL per 96 well plate o IBMX (heat the 500 mM/DMSO stock in the water bath and pipette up and down until completely dissolved) • Prepare the 5 pM forskolin stimulation medium • Prepare the ligand dilutions • Aspirate the serum-free medium observing the precautions mentioned above • Immediately fill the empty wells with base, forskolin or ligand dilution media, accordingly to the design of the experiment. Return the plates in the CO 2 incubator for o/n incubation

Step 6: Start o f SEAP assay

• Remove the 96 well plates from the incubator • Centrifuge the plates (use the swinging bucket centrifuge in room 321) at ca 3000 rpm for 20 min • Transfer the medium to a clean 96 well plate (no need to be sterile) • Seal the plates with culture medium to prevent evaporation (the Press'n Seal wrapping foil seems to work fine) • Incubate the sealed plates at 65 °C for 20 min • Place the plates onto a cool surface then heat the membrane to remove the water condensed on it • Transfer 50 pL 2X DEA SEAP buffer to each well of a fresh, clean 96 well plate • Add 50 pL of culture medium to the SEAP buffer in the new plate Mix briefly the plate on the vortexer at the minimum speed setting Immediately start the chronometer for this plate • Take the first OD reading for this plate on the plate reader at 405 nm • Store the plates at r.t. in the dark (or use tin foil) between readings

Data Analysis

The result of OD readings at different time intervals is the progress curve of the enzymatic reaction (OD vs time) The initial slope of the curve (time'1) is proportional with the concentration of enzyme (SEAP) in the medium sample which in turn describes the degree of activation of the cAMP dependent signal transduction pathway under the corresponding experimental conditions The initial rate in the absence of a treatment (ligand or Fsk) for cells co transfected with the blank vector corresponds to the activation of the STP due to accumulation of cAMP due to normal metabolic activity of the cell (basal activity Vb)

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The initial rate in the absence of a treatment (ligand or Fsk) for cells co transfected with the receptor construct corresponds to the activation of the STP due to accumulation of cAMP due to normal metabolic activity of the cell plus the constitutive activity of the receptor (Vb + Vc) The initial rate for the cultures exposed to a stimulus (Fsk or ligand) corresponds to the sum between basal, constitutive and stimulus induced STP activation (Vb + Vc + Vf or Vb + Vc + Vdi) Vdi is the activation of the STP due to the treatment with the concentration (dose) /' of ligand Calculation of the normalized constitutive activity: VNc = Vc/Vf = ( (Vb+Vc)-Vb) / ( (Vb+Vc+Vf) - (Vb+Vc) ) Calculation of the normalized ligand induced actuvity: VNdi = Vdi/Vf = ( (Vb+Vc)-Vb)/( (Vb+Vc+Vf) - (Vb+Vc))

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX C. Oligonucleotides Used in the Study of Lamprey GpH-R I and II

Table 5. Oligonucleotides used in the study of lamprey GpH-R I and II. N= any nucleotide, Y=C or T, R=G or A, D=G, A or T.

Name Sequence Comments

IGpH-R I primers G H R S 1 128f TTYAAYCCSTGCGARGAYATCATGGGMTAYGA GLYCHORMONER element 1 G H R S 2 256 f TAYAARCTSACSGTSCCSCGCTTYCTSATGTG GLYCHORMONER element 2 G H R S 7 5 1 2f ATCACSGTSACSAAYWSCAARATCCTSCTSGT GLYCHORMONER element 7 C 2 P 6 f CATCAGCAGCTACTCCAAGGTGAGCATTTG Gene specific primer for IGpH-R I RT-PCR assay C 1 P 6 f CTCCTCAT CAT CCT GACG AGCCACTACAAG Gene specific primer for IGpH-R II RT-PCR assay 5proxB CGACGTGGACTATCCATGAACGCAAAGCAGTGGTATCAA Step-Out RACE 5' adaptor 1 [Matz2003] CGCAGAGT Udist TCGAGCGGCCGCCCGGGCAGGTCGACGTGGACTATCCA Step-Out RACE 5' adaptor 2 [Matz2003] TGAACGCA IGpHRI_TOPOf CACCATGGGTTGGGAGCACCGTAGGACGTC 5' primer for cloning the IGpH-R I cds for functional studies G H R S 7 5 1 2r AGSAGGATYTTGSWRTTSGTSACSGTGATSAG GLYCHORMONER element 7 G H R S 8 512 r RAAYTT G S WSAG SAGG ATRAARAART CGCGGC GLYCHORMONER element 8 G H R S 3 144r CAKCCVGCKCCSGTCTGCCARTCGATVGCGTG GLYCHORMONER element 3 C 2 P 2r AAGCTCTTGGTGAAGATGGCGTAGAGTAAGGG Gene specific primer for IGpH-R I RT-PCR assay C 1 P 4 r GTTACGCACGGTGATGTAAATGTGCACGTAG Gene specific primer for IGpH-R II RT-PCR assay

C D S III AFTCTAGAGGCCGAGGCGGCCGACATG 1 FI ! 1111I'TTTT Clontech SMART cDNA library construction kit 1 1 1 1 111 1 1 1 1 1 1 1 1 1TNN IGpHRI_TOPOr CGTACGGCGGTGTAATTGAGCCGCGTTACG 3' primer for cloning the IGpH-R I cds for functional studies G SP6r1 CCAATCGATAGCGTGGTTGTGATACTC Gene specific primer for second stage 5' SO-RACE SP 1C 2r1 GCATGGTGTAGATGATGGTGTGCCA Gene specific primer for second stage 5' SO-RACE G SP7r1 ATGCTC ACCTTGGAGTAGCTGCT GAT G Gene specific primer for second stage 5' SO-RACE SP1 C 2r2 TAGCTCGGGTTCCTAACCGTTGAGTAGAT Gene specific primer for second stage 5' SO-RACE C 2 P 2r AAGCTCTTGGTGAAGATGGCGTAGAGTAAGGG Gene specific primer for first stage 5' SO-RACE IGpH-R II primers

P1_lgphrii_ex6_f AACGCGTTTGAAGGCCTGAGC IGpH-R II exon 6 forward primer (fragment S4) P2_lgphrii_ex6_f AG ATGGCT G ACAAT CT C AACAT GCC IGpH-R II exon 6 forward primer (fragment S4) P1_lgphrii_ex6_r GGCATGTTGAGATTGTCAGCCATC IGpH-R II exon 6 reverse primer (SO-RACE, S3) P2_lgphrii_ex6_r AAACGCGTTGGCAGGAATCAGG IGpH-R II exon 6 reverse primer (SO-RACE, S3) P3_lgphrii_ex6_r TTCAAACGCGTTGGCAGGAATC IGpH-R II exon 6 reverse primer (SO-RACE, S3)

IGpHRI l_cds5_Kpnl ATAATAGGTACCATGGCGGTTGGAGGATCGGCGCTGAC IGpH-R II CDS 5' primer, inserts Kpn I cutting site Pf_flag_lgphriied GACTACAAGGACGATGACGATAAGTGGCTGTGTCCGGCC IGpH-R II extracellular domain forward primer AATTGCCGATG

IGpHRII_cds5_TOPO CACCATGGCGGTTGGAGGATCGGCGCTGAC IG pH -R II C D S 5' prim er for T O P O cloning IGpHRII cdsS Kpnl T CACCGGTACCATGGCGGTTGGAGGATCGGCGCTGAC IGpH-R II CDS 5' primer for mixed TOPO/Kpn I OPO cloning IGpHRII_cds3 CGCTCACAGGGCGCACCGCACGCATGTG IGpH-R II CDS 3' primer (TOPO cloning) IGpHRII_cds3_Xhol TATTATCTCGAGTCACAGGGCGCACCGCACGCATGTG IGpH-R II CDS 3' primer (sticky end, Xho I cloning)

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APPENDIX D. Chemical Communication in Vertebrates

One of the dimensions of the mechanisms of homeostasis maintenance in the multicellular organisms is the compartmentalization of their different physiological functions. A multicellular organism is not an amorphous collection of cells but an assembly of specialized tissues and organs each of them exhibiting a specific set of metabolic and structural characteristics. These anatomic-functional units are involved in particular segments of the interactions between the external and internal factors leading ultimately to the self-preservation and reproduction of the whole system. These units can be described as independent systems, but the essence of their role is the communication between them and correlation of their parameters in response to external and internal changes. All physiological functions of an organism are controlled by an intricate network of chemical signals. This transfer of information and regulation of the functions of physiological compartments is mediated by three independent but overlapping physiological subsystems: the endocrine system, the nervous system and the immune system. A a wide range of the small molecular and proteic content of the internal medium has an information carrying function. These molecules are the messengers which allow different organs and tissues to communicate with each other, to transmit the parameters of their status over the anatomic boundaries inside the organism. They are classified as a special class of functional molecules called 'hormones'. Endocrinology is the scientific discipline studying the hormones and the processes and structures associated with their secretion, transport and action. The concept of chemical factors acting as regulatory factors in physiology is a relatively recent development in the study of physiology. Although western medicine acknowledged the role of the 'internal medium' in normal and pathological conditions it was only attributed to a passive role in the transfer of nutrients from blood to tissues. At the beginning of the 19th century, Claude Bernard revived Galen's old humoral theory in the concept of 'internal medium'; also by extensive experimentation he showed evidence for the existence of the 'internal secretion'. However, in the 19th century the nervous system was considered the only basis for the regulatory mechanisms, due to the success of Pavlov's experiments on digestive reflexes in dogs. In 1891 Murray successfully treated a myxoedematous patient with an extract of thyroid and in 1894 Oliver and Schaefer described epinephrine. It was only in the dawn of the 20th century, in 1903 when Bayliss and Starling showed that an extract of duodenum injected into a dog induced a pancreatic secretion not attributable to a nervous causation. Later (1905) Starling coined the term of 'hormone' for the chemical factors involved in regulation of physiological processes ([159] [160], [161]). Endocrinology as a scientific discipline in its own rights was just being born. The central paradigm in endocrinology relies on chemical factors (hormones) acting on a distant target, at the level of a specific receptor. A hormone is defined as any substance formed in very small amounts in one specialized organ or group of cells and carried (usually via the bloodstream) to

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. another organ or group of cells, in the same organism, upon which it has a specific regulatory action triggered by its binding to a specific receptor in that tissue. Regulators of physiological processes in vertebrates belong to a wide range of different types of molecules. According to their chemical nature they are classified as peptide, amine, steroid and eicosanoid derived hormones. In the last decades, a few more products of metabolism (e.g. nitric oxide) were found to be involved in actions which would qualify them as hormones. The traditional paradigm of hormones is that they are active chemicals being synthesized in specialized glands, transported in the bloodstream to the target tissue where they exert their actions. This view has been challenged by the finding that many hormones have also a more confined area of action, some of them acting on cells of different type immediately surrounding the secretory cells (paracrine effect) or they can even modulate the metabolism of the secretory cells themselves (autocrine effect). This concept of hormones acting locally, as well as distantly, acting chemical regulators is reshaping the understanding of endocrine control from an endocrine pathway/endocrine axis centered paradigm to a multileveled, ubiquitous and finely structured network of informational channels. A receptor is a protein acting as a transducer of information between different physiological and/or metabolic compartments. The existence of receptors in animal tissues was derived historically from observations made on the selective properties of the interactions between tissues and various chemical agents (dies, drugs, etc). The distributive explanation of this behavior was initially predominant: the selectivity is the result of the intrinsic properties of the chemical agent, determining a specific pattern of interaction with different tissues. A first contribution was by Ehrlich (1854-1915) who hypothesized that the selectivity of action of chemical agents is also conditioned by the existence of tissue specific factors which need to interact specifically with the chemical agent in order for the tissue specific behavior to become manifest (“Corpora non agunt nixi fixata” - the agents need to be bound first in order to act). Later, in 1948 Ahlquist showed that epinephrine, norepinephrine, and isoproterenol induced two different types of responses in the smooth muscle (contraction and relaxation) and the potency of these drugs was different and dependent on the type of response. This was not in agreement with the distributive explanation of the drug/tissue interaction and could not be explained by the absence of specific interactions between the drug and a tissue intrinsic factor. Moreover, it has become evident that the degree of occupancy of the tissue receptors can not explain the differential effect of the drugs unless existence of different receptors, associated with different types of responses was accepted. Discovery of antibodies capable of acting as insulin agonists suggested that the signal transduction capability is a property of the receptor and not of the ligand. It has also been shown that some pathological conditions originate in the presence of deficient receptors capable of activating the signal transduction in the absence of the ligand (constitutive activity). The modern concept of receptor is that of a protein integrated in the cell membrane (cell surface receptor) or part of the cytoplasm (nuclear receptor)

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. acting as transducers of information between different physiological/metabolic compartments. The cell surface receptors are confined to the plasma membrane and transmit the signal to the intracellular machinery via a chain of regulatory interactions usually referred to as a 'signal transduction pathways'. The 'nuclear receptors' in their inactive form are located in the cytoplasm or nucleus, complexed with other proteins, usually heat shock proteins (HSP). They dissociate and migrate into the nucleus upon binding of the a small, hydrophobic hormone (steroid, thyroid hormones, vitamin D or a retinoic acid derivative). Inside the nucleus they act as transcription modulators directly by binding to specific motifs (cis-regulatory elements) in the DNA sequence ('ligand activated transcription factors') or indirectly by binding to other transcription regulatory proteins. This results in the activation of the transcription of the corresponding genes [162]. There is usually a physical separation between compartments interacting via receptor mediation but this is not a requirement; more importantly, hormone receptors connect physiological niches which are functionally separated. The Hvpothalamic-Pituitarv-Peripheral Gland (HPPG) Endocrine Control Hierarchy. The hormone/receptor pairs of the endocrine systems of vertebrates are coupled into chains of regulatory interactions which in effect connect the internal and external stimuli to metabolic and physiological capabilities of response of the organism. The organization of the hypothalamic-pituitary- endocrine axes in vertebrates has been extensively studied and is well understood ([163]). At the top level, the activity of the endocrine systems are controlled by the products of secretion of the neurons in neuro-secretory nuclei located in the hypothalamus and pre-optic area of the brain [164]. Neuropeptides released from the hypothalamus in blood reach the anterior hypophysis via the hypothalamic/pituitary portal system. Here they activate receptors in specific fields of secretory cells which release their products (hypophyseal trophic hormones) into the general circulation. Circulating hormones will bind to specific receptors expressed in different target tissues of the organism starting a new chain of effects. Some of the products of activation of target tissues released into the general circulation will activate receptors located in the pituitary and/or hypothalamus closing the feed-back connection. This regulatory loop controls directly or indirectly many endocrine systems in Gnathostomes (jawed vertebrates). In spite of the vast adaptive variations in morphology, physiological rhythms, life cycles and behaviors, all vertebrates studied so far follow this same basic design of their endocrine control. Located on the ventral part of the central nervous system, the hypothalamus, together with the pre-optic area is the site of a number of neuro-secretory nuclei which have been shown at the end of the first half of the past century to be the site of synthesis of a large number of products of secretion responsible for the modulation of the activity of the pituitary gland. The hypothalamus is a very old component of the vertebrate brain. It is present in the central nervous system of all vertebrates, including hagfish and lamprey. Its morphology is also similar in all vertebrates.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Thyroid hormones

HO HO

'NHi' r ’HO 1 1 N H , HO Thyroxine (T4) 5,3'-triiodothyronine (T3)

Sex steroids OH HO

HO Estradiol Progesterone Testosterone

Figure D.29. Thyroid hormones (T3J4) and gonadal steroid hormones.

The pituitary gland (hypophysis ) is a small secretory organ located on the ventral part of the brain, in close proximity to the hypothalamus. It was traditionally considered the 'master gland' and is divided into the anterior pituitary (adenohypophisis) and posterior pituitary (neurohypophysis) The hypophyseal divisions have different developmental origins and different functional characteristics. The anterior pituitary is larger and has an ectodermal origin resulting from a projection of the roof of the mouth (Rathke's pouch) protruding upwards during embryogenesis. The posterior pituitary results from a down growth of the diencephalon which contacts Rathke's pouch and remains attached to the hypothalamus through the infundibular stalk. The posterior pituitary acts as a neurohemal organ, its hormonal releases, oxytocin and arginin vasopressin (AVP or antidiuretic hormone ADH) are the products of the secretory neurons in the hypothalamus, and are transported to the neurohypophysis through their axonal endings in the infundibular tract. The anterior pituitary (adenohypophysis) is a true gland and contains five major types of secretory cells: gonadotrophs, thyrotrophs, corticotrophs, somatotrophs, melanotrophs and lactotrophs. Their hormonal secretion products are the gonadotropins (LH and FSH), thyrotropin (TSH), adenocorticotropin (ACTH), somatotropin (growth hormone, GH), melanocyte stimulating hormone (MSH) and prolactin (PRL) respectively. All of them are peptide hormones and their release is induced by the hypothalamic releasing hormones transported to the anterior pituitary via the hypothalamic-pituitary portal system.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Hormones of the Hypothalamic-Pituitary-Gonadal (HPG) and Hypothalamic- Pituitary-Thyroid (HPT) Axes. The HPG endocrine axis is defined as the assembly of hormones, their receptors and the anatomic-functional units implied in the regulation of the development and function of the reproductive function in vertebrates. Similarly, the HPT axis is defined as the assembly of hormones, receptors and anatomic- functional units implied in the regulation of the development and function of the thyroid gland. The functional selectivity of these two similar endocrine control pathways is ultimately determined by the specificity of interaction between the pituitary hormones and and their cognate receptors as well as by the pattern of expression of the receptors [135]. The simplest description of the HPG and HPT axes involves three levels of control: the hypothalamic (neural) level, the pituitary level and peripheral gland level. The peripheral level: Thyroid and gonadal steroid hormones are small hydrophobic hormones that act through paracrine and endocrine mechanisms, bind to specific nuclear receptors and trigger pleiotropic effects at both the local and organismal level. Thyroid hormones (Figure D.29) are small hydrophobic hormones and called thyroxine and triiodothyronine. They are responsible primarily for the regulation of cellular energy metabolism. In different organisms, the thyroid hormones are important factors controlling various stages of development. In amphibians for example, thyroid hormone secretion is associated with the onset of metamorphosis. The pituitary level: Gonadotropin and thyrotropin (glycoprotein) hormones are heterodimeric proteins released into the general circulation from adeno hypophysis specific cells in response to hypothalamic releasing hormones pulsatory stimulation. Glycoprotein hormones are tropic proteins transported through the bloodstream to the target organs (gonads and thyroid gland). They are one of the largest and most complicated hormone ligands known at this point. Depending on their target tissue they are classified into two groups the gonadotropins and the thyrotropin hormones. In all vertebrates at least two types of gonadotropins are found: the follicle stimulating hormone (follitropin, FSH) and luteinizing hormone (lutropin, LH) and one thyrotropin hormone. In primates there is a third type of gonadotropin, chorionic gonadotropin (CG), encoded by a distinct duplicated gene, secreted not from the pituitary but from the placenta and showing the highest binding affinity for the same receptor as LH. The pituitary tropic hormones, luteinizing hormone (LH) and follicle stimulating hormone (FSH), collectively known as gonadotropins, are synthesized and released into the blood stream in response to the action of the hypothalamic decapeptide (gonadotropin releasing hormone, GnRH). Once in the bloodstream they travel to the target organs where they stimulate a cascade of metabolic processes which result mainly in the synthesis of steroid hormones in the gonads. In all vertebrates, the activity of the HPG follows a cyclic pattern with the amplitude of oscillations varying largely among taxa. This requires a very finely tuned cooperation among different control systems [165], [166].

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Pituitary glycoprotein hormones are ca. 20 kDa heterodimeric proteins composed of two similar subunits, both of them members of the cystine-knot superfamily of growth factors (CKGF) which includes other hormones like GH, cytokines, growth factors etc. This family of functional proteins has a very diverse composition and the similarity scores among the members of its subfamilies is low [167]. However, all of them are characterized by the presence of a specific Cys motif ('cystine knot' motif). The Cys residues in this motif are connected by disulfide bridges which generate a characteristic 'knot' which gives the name of the protein superfamily [23], [17], [56], [168]. Glycoprotein hormone subunits are non covalently linked and designated alpha and beta respectively. Alpha subunits are common to all pituitary glycoprotein hormones. Beta subunits on the other hand are characteristic to each type of hormone and are considered responsible for the specificity of binding of the hormone to the receptors. Gonadotropins and thyrotropin are secreted from specific cell types in the anterior pituitary, the gonadotrophs and thyrotrophs respectively [91]. Lengths of the glycoprotein hormone beta chains vary between 130 and 150 amino-acid residues. The alpha chains are usually shorter, under 120 amino acid residues and in some species (rabbit, whale) they are under 100 residues. The isoelectric points of the alpha chains are slightly basic (IP=7.5-9) while beta chains have isoelectric points varying in different species between acidic (IP=4.1) to slightly basic (IP=8). Heterogeneity of oligosaccharide composition in mammalian glycoprotein hormones has raised the question of its impact on the function of the hormones [169]. The hypothalamic level: Gonadotropin and thyrotropin releasing hormones are small neuropeptides secreted from hypothalamic nuclei and in most vertebrates reach the adenohypophysis via a short, local and fast circulatory subsystem — the hypothalamic-pituitary portal system. The gonadotropin releasing hormones (GnRH) are decapeptides secreted from the hypothalamus in a pulsatile manner. Most vertebrates express two forms of GnRH. Usually only one of them is active in the HPG axis and is expressed predominantly in the hypothalamus. The other form has a more diffuse pattern of tissue expression and its role is not very well established. The gonadotropin releasing hormone forms are identified by the name of the species in which it has been initially described. The 'mammalian' GnRH is in fact part of the HPG axes in most tetrapods, while 'chicken' GnRH-ll is common to all vertebrates and usually does not participate in the control of reproduction. A separate situation can be found in Teleosts where a a number of species characteristic GnRH forms have been isolated and shown to be responsible for the control of reproduction. Finally, in the sea lamprey there are at least two forms of GnRH (lamprey GnRH I and lamprey GnRH III), and their relative contribution to the reproductive physiology of this animal is still being investigated. The signal transduction pathways activation by GnRH in gonadotrophs in mammals is highly sensitive to the frequency of the GnRH stimulatory pulses. Correspondingly, the effects of GnRH are different having a stimulatory effect on LH and FSH when administered in pulses or an inhibitory effect on LH and FSH biogenesis when it is applied in a sustained manner. The

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. receptors for gonadotropin releasing hormone belong to the G-Protein Coupled Receptor superfamily. The ligand GnRH binds to the extracellular loops of the 7 transmembrane domain inducing conformational changes which activate the intracellular signal transduction pathways. The GnRH receptor is usually described as an activator of IP3/DAG mediated signaling via interaction with the Gq/11 G-protein. However, it has been well established that the cAMP dependent signaling is also important for the proper functioning of the gonadotrophs and moreover that an increase in the cAMP concentration can be detected in vitro following stimulation with GnRH. The mechanisms by which GnRH induces cAMP synthesis and activation of genes under control of the CRE cis-regulatory element is not yet well known and subject to active investigation ([170]). The TRH (thyrotropin releasing hormone) tripeptide was the first hypothalamic releasing factor isolated [171]. Extended endocrine control: a much larger number of factors contribute to the function of the HPG/T axes. Recently, new putative components of the HPT axis have been identified ([172]). A homology search in the human genome sequences at GenBank retrieved a new dimeric glycoprotein with characteristics common with the Cys- knot group of growth factors and with the pituitary glycoprotein hormones TSH, LH and FSH, in particular ([173]). Although its similarity is low when compared with the pituitary GpHs, this new molecule was a powerful activator of the thyrotropin receptor so it was termed thyrostimulin. The two subunits of thyrostimulin (A2 and B5 in author nomenclature) were shown to be expressed in a many more types of tissues than the pituitary TSH, including thyroid follicles, pituitary, ovary and brain. Its normal and pathological physiology roles are yet to be clarified but it is assumed that it will mostly act through a paracrine mechanism, particularly due to the fact that its expression was found in tissues were the expression of the thyrotropin receptors has already been established (brain, thyroid, heart and oviduct) ([172]).

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