The Mechanism of Lactogen Receptor Binding by Human Prolactin

Home , Prolactin receptor

THE MECHANISM OF LACTOGEN RECEPTOR BINDING BY HUMAN PROLACTIN

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Umasundari Sivaprasad, M.S.

* * * * *

The Ohio State University 2003

Dissertation Committee:

Professor Charles L. Brooks, Adviser Approved by

Professor Caroline Breitenberger

Professor Russ Hille

Professor George Marzluf Adviser The Ohio State Biochemistry Program

ABSTRACT

Communication between multiple binding sites in a macromolecule has been demonstrated to occur through conformation changes propagated through regions of the molecules that do not constitute the binding interfaces.

This work has examined whether two receptor-binding sites on a hormone are functionally coupled.

Prolactin, a reproductive hormone that belongs to the lactogenic hormone family with growth hormone and placental lactogen, binds to two prolactin receptors in a sequential manner, the first receptor binding at Site 1 of the hormone followed by the second receptor binding at Site 2. The detailed mechanics of receptor binding however are unclear. In the work presented here, the mechanism by which the extracellular domain of the human prolactin receptor binds to human prolactin has been investigated using surface plasmon resonance technology where prolactin is coupled to a dextran surface using bulky coupling chemistries that reside in, and block either Site 1 or Site 2. When the receptor is passed over the protein with Site

2 blocked by the coupling linkage, the receptor binds to the hormone, indicating that Site 1 function is independent of Site 2. However, if Site 1 is blocked by the coupling linkage, no receptor binding can be measured,

ii suggesting that receptor binding at Site 2 is dependent on receptor occupancy of Site 1. Kinetic analysis of binding site mutants has further supported this hypothesis since corruption of Site 1 dramatically reduces the amount of receptor bound to the hormone. Using site-directed mutagenesis, a contiguous set of hydrophobic residues in hPRL has been identified, whose mutation reduces the Site 2 activity of hPRL and is proposed to transmit a conformational change initiated by Site 1 binding to organize and create a functional chemical geometry within Site 2. We propose that binding of one receptor to hPRL induces a conformation change in the hormone, which is transmitted by the hydrophobic residues and activates the second receptor- binding site.

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Dedicated to my parents

ACKNOWLEDGMENTS

I wish to thank my adviser Dr. Charles L. Brooks for his encouragement, guidance and patience. The numerous discussions we had over the years have significantly impacted my scientific thinking and outlook.

This work would not be possible without his support.

I would also like to thank Dr. Karen Duda, for her helpful discussions and guidance when I first joined the lab. I am grateful to Dr. Colleen Almgren,

Toni Hoepf, Jeffrey Voorhees, Scott McCardle and other lab members for their friendship and encouragement.

I would like to thank Dr. Kathleen A. Hayes for her help with statistical analysis of the kinetic data, Gordon Renkes for his help with CD spectroscopy, and the staff at the Campus Chemical Instrumentation Center (CCIC), particularly Dr. Kari Green-Church for their help with performing the mass spectrometry and data analysis.

And last but certainly not the least, I would like to thank my sister, Usha

Sivaprasad and my friend Dr. Melinda Butsch Kovacic for their support and tremendous patience with me.

VITA

August 26, 1973………………………Born, Chennai, India

1993……………………………………B.S. Microbiology/Biochemistry University of Mumbai, India

1995……………………………………M.S. Biochemistry University of Mumbai, India

1995 –1996……………………………Product Manager, Protec Ltd. A Division of Cipla Ltd., Mumbai, India

1996 – present………………………. Graduate Research Associate The Ohio State University

PUBLICATIONS

Hai, T., Wolfgang, C. W., Marsee, D. K., Allen, A. E., Sivaprasad, U., (1999). ATF3 and Stress Responses (Review). Gene Expression 7, 321-335.

FIELDS OF STUDY

Major Field: Biochemistry

TABLE OF CONTENTS

Abstract………………………………………………………………………..ii

Dedication……………………………………………………………………..iv

Acknowledgments……………………………………………………………v

Vita…………………………………………………………………………….vi

List of Tables…………………………………………………………………xi

List of Figures………………………………………………………………..xii

Chapters:

1. Introduction…………………………………………………………………1

1.1 Overview of the project…………………………………………...... 1

1.2 Theories on the functional coupling of ligand binding sites………..1

1.3 Background and physiology of hPRL………………………………...3

1.4 Structure-function studies on hPRL…………………………………..8

1.5 The prolactin receptor…………………………………………………18

2. Materials and Methods…………………………………………………...24

2.1 The pT7-7 phagemid…………………………………………………24

2.2 Primer design for site-directed mutagenesis………………………25

2.3 Site-directed mutagenesis…………………………………………...25

2.4 Expression, folding and purification of proteins…………………...26

vii 2.5 Characterization of recombinant proteins………………………….28

2.5.1 Polyacrylamide Gel Electrophoresis (SDS-PAGE).………...28

2.5.2 UV Absorption Spectroscopy………………………………….28

2.5.3 Fluorescence Spectroscopy…………………………………..29

2.5.4 Circular Dichroism (CD) Spectroscopy………………………30

2.5.5 Mass Spectrometry…………………………………………….30

2.6 Lactogenic Bioassays………………………………………………..31

2.7 Surface Plasmon Resonance Experiments………………………..32

2.7.1 Immobilization of the hormones on the dextran-coated

chip surface……………………………………………………..33

2.7.2 Mass transport limitation experiments……………………….35

2.7.3 Equilibrium binding experiments……………………………..35

2.7.4 Binding of hPRLbp to the hPRL mutants……………………36

2.7.5 Evaluation of kinetic data……………………………………..36

2.8 Fluorescence Resonance Energy Transfer (FRET)……………...37

3. Mechanism of Ordered Lactogen Receptor Binding…………………..39

3.1 Introduction……………………………………………………………39

3.2 Results…………………………………………………………………42

3.2.1 Characterization of the Recombinant Proteins……………...42

3.2.2 Effect of blocking Site 1 or Site 2 on receptor

binding capacity………………………………………………..53

3.2.3 Kinetics of receptor binding to M158C and G129C………...54

viii 3.3 Discussion……………………………………………………………..61

4. Effect of Corruption of Site 1 and Site 2 on Receptor Binding……….64

4.1 Introduction……………………………………………………………64

4.2 Results………………………………………………………………...65

4.2.1 Characterization of the mutants……………………………...65

4.2.2 Receptor binding to hPRL mutants with increasing

disruption of Site 1…………………………………………….71

4.2.3 Receptor binding to a mutant with a corrupt Site 2………...75

4.3 Discussion…………………………………………………………….77

5. Conformation Change Induced by Receptor Binding…………………79

5.1 Introduction……………………………………………………………79

5.2 Results………………………………………………………………...81

5.2.1 Absorption spectra of proteins labeled with CPM……….....81

5.2.2 Fluorescence Resonance Energy Transfer (FRET)………..82

5.3 Discussion……………………………………………………………..88

6. Identification of Residues Critical for Prolactin Activity……………….90

6.1 Introduction……………………………………………………………90

6.2 Results………………………………………………………………...94

6.2.1 Characterization of the mutants in helix 2 and helix 4……..94

6.2.2 Biological activity of the helix 2 and helix 4 mutants……….96

6.2.3 Characterization of the mini-helix 1 mutants………………..97

6.2.4 Biological activity of the mini-helix 1 mutants……………….98

ix 6.3 Discussion……………………………………………………………110

7. Discussion and Perspectives…………………………………………....118

List of References……………………………………………………………124

LIST OF TABLES

Table Page

1.1 Amino acids mutated in human prolactin and the effects on

biological activity and receptor binding …………..……………………14

3.1 Rate and equilibrium constants for receptor binding to the

cysteine mutants …………………………………………………………60

4.1 ED50 values of the various hPRL mutants……………………………..71

4.2 Rate and equilibrium constants for hPRL binding site mutants……..74

5.1 Increases in fluorescence intensity of CPM in labeled hormones

upon receptor binding……………………………………………………87

6.1 ED50 values helix 2 and helix 4 mutants………………..…………….104

LIST OF FIGURES

Figure Page

1.1 Model of human prolactin………………………………………….....11

1.2 Location of key residues in the putative Site 1 and Site 2………..17

1.3 The extracellular domain of the prolactin receptor…………………22

3.1 Location of residues in hPRL mutated to cysteine………………...40

3.2 SDS-PAGE gels of recombinant protein preparations…………….46

3.3 Spectroscopic characterization of the cysteine mutants…………..47

3.4 UV, Fluorescence and CD spectroscopy of hPRLbp……………....49

3.5 Lactogenic bioassay of the binding site cysteine mutants…………50

3.6 Competitive bioassay of recombinant hPRLbp……………………..51

3.7 UV spectroscopy of samples treated with DTT……………………..52

3.8 Equilibrium binding of hPRLbp to the cysteine mutants…………...56

3.9 Comparison of hPRLbp binding to M158C and I146C……………..57

3.10 Fitting of the receptor binding curves to the cysteine mutants…….58

4.1 SDS-PAGE gels of the hPRL mutants……………………………….67

4.2 Spectroscopic analysis of the hPRL mutants……………………….68

4.3 Lactogenic bioassay of the hPRL mutants…………………………..70

4.4 hPRLbp binding to the Site 1 mutants……………………………….73

4.5 Binding of hPRLbp to G129R/M158C compared to M158C………76 xii 5.1 Absorption spectra comparing labeled and unlabeled proteins…..84

5.2 Fluorescence spectra of labeled proteins mixed with increasing

receptor concentrations……………………………………………….85

5.3 Competition experiment using excess unlabeled hPRL…………...86

6.1 Comparison of the primary amino acid sequence of hPRL

and hGH…………………………………………………………………93

6.2 SDS-PAGE gels of mutants in helix 2 and helix 4………………….99

6.3 Absorption spectra of helix 2 and helix 4 mutants………………...100

6.4 Fluorescence spectra of helix 2 and helix 4 mutants……………..101

6.5 CD spectra of helix 2 and helix 4 mutants………………………….102

6.6 Lactogenic assay of helix 2 and helix 4 mutants…………………..103

6.7 SDS-PAGE gels of mini-helix 1 mutants……………………………105

6.8 Absorption spectra of mini-helix 1 mutants…………………………106

6.9 Fluorescence spectra of mini-helix 1 mutants……………………...107

6.10 CD spectra of mini-helix 1 mutants………………………………….108

6.11 Lactogenic bioassay of mini-helix 1 mutants……………………….109

6.12 Position of all the residues mutated in mini-helix 1, helix 2

and helix 4……………………………………………………………...114

6.13 Articulation between hydrophobic residues in the hPRL model.....115

7.1 Comparison of the NMR structure and model of hPRL……………123

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CHAPTER 1

INTRODUCTION

1.1 Overview of the Project

Numerous studies have demonstrated that conformation changes are induced in the presence or absence of allosteric modulators, regulating the ligand binding properties of enzymes and other proteins. The goal of this work was to determine whether this observation could be extended to receptor binding sites on hormones. In the work described here, the interaction of human prolactin (hPRL) with the extra-cellular domain of the prolactin receptor

(hPRLbp) has been used as a model system to study the mechanism of receptor binding. The evidence presented here suggests that the sequential binding of the two receptors to prolactin is a result of a restructuring of the second receptor binding site occurring after the first receptor binds to the first binding site, supporting an induced-fit model.

1.2 Theories on the functional coupling of ligand-binding sites

A significant amount of the early work done on allosteric regulation of ligand binding was performed on hemoglobin (Hb)(1). Hb has four oxygen 1 binding sites and the binding curve suggests cooperativity between the sites.

However the mechanism for cooperativity was unclear. In 1948, Jeffries

Wyman proposed that the ligand-binding sites on Hb are “functionally linked”

(2). He proposed that there was equilibrium between two states of Hb with oxygen having a higher affinity for one of the states. This idea was incorporated into a “two-state model” proposed by Monod, Wyman and

Changeux (3). The limitation of this model was that it assumed symmetry between the various binding sites, limiting the number of transition states in

Hb. In 1966, Koshland, Nemethy and Filmer proposed another model that states that progress from the low O2 affinity state to the high O2 affinity state is a sequential process (4). Binding of oxygen to the first subunit of Hb causes a conformation change in the subunit. This conformation change is transmitted to the second subunit that can then bind another oxygen. This “induced-fit” model suggests that substrate binding to an enzyme could elicit a conformation change resulting in a more favorable spatial rearrangement of atoms in the ligand-binding site. The “induced-fit” model can describe the binding mechanism of phosphofructokinase (5), glycogen phosphorylase (5), and chloroplast ATP synthase (6) among others. This induced-fit mechanism has also been used to describe hormone receptor interactions, although most of the studies have been done on conformation changes in the receptor.

These include conformation changes in the insulin receptor induced by insulin binding (7), and agonist induced conformation change in the β2 adrenergic

2 receptor (8). Recent studies have shown that there is a discreet hydrophobic motif in human growth hormone, distal to the two receptor binding sites, that transmits a conformation change in the hormone allowing the second receptor to bind (110).

Prolactin, a lactogenic hormone, is believed to bind two prolactin receptors in a sequential manner (9,10). However the detailed mechanism is not known. One possible mechanism could be that the two binding sites have different affinities for the receptor. Alternatively, receptor binding at the first site could induce a conformation change in the hormone, spatially reorganizing the atoms at the second site allowing the second receptor to bind – an

“induced-fit” model. The goal of the work described here is to clarify which mechanism best describes prolactin receptor binding by the lactogenic hormone.

1.3 Background and Physiology of hPRL

Riddle and coworkers first purified prolactin in 1933 from dissected anterior pituitary lobes of cows, sheep and pigs (11). Over the next 50 years numerous attempts were made to purify human prolactin. Pituitary homogenates contained prolactin-like activity but preparations yielded only growth hormone. Human growth hormone was purified in 1956 (12) and showed prolactin-like activities. For many years it was thought that growth hormone had evolved to serve dual functions in human cells. The functional

3 similarity between human growth hormone and prolactin (unlike other species) and the 100-fold excess concentration of growth hormone in human pituitaries made separation of prolactin very difficult (reviewed in (13)). Then, in the early

1970’s, two independent groups purified human prolactin and demonstrated that it was indeed independent of growth hormone (14) (15). Although the work presented here primarily focuses on human prolactin, a lot of work has been done on prolactin from other species. The significant homology between prolactin from various species has allowed some extrapolation of work between different species. Work done on PRL from other species that is relevant to the work described here will be included where appropriate.

The human prolactin (hPRL) gene is located as a single copy on chromosome 6. The gene is made up of six exons and is more than 15kb in length (reviewed in (16)). In the pituitary, the transcription begins with exon

1b. However in extrapituitary cells, the transcription begins at exon 1a resulting in a transcript with an additional 150bp. This additional length lies in the 5’ untranslated region (5’ UTR) of the transcript. Both transcripts are translated to give protein of the same length. Prolactin is produced as a pre- hormone, 227 amino acids long. The 28 amino acid signal peptide is cleaved upon entering the endoplasmic reticulum, resulting in a secreted protein 199 amino acids in length and a molecular weight of approximately 23,000 Daltons

(reviewed in (17) and references therein).

4 Expression of pituitary and extrapituitary prolactin is differentially regulated (16). In the pituitary, prolactin gene transcription is controlled by a proximal promoter approximately 2-2.5 kb in length that requires the transcription factor Pit-1. Two regions are recognized in the promoter – a proximal promoter and a distal enhancer. In extrapituitary cells, a distal promoter about 5800 bp upstream of the start site controls transcription. This promoter is silenced in the pituitary.

Prolactin undergoes various posttranslational modifications (reviewed in

(18,19)). Some of the modifications include phosphorylation, glycosylation, deamidation, proteolytic cleavage and conjugation with other proteins.

Phosphorylation of rPRL at Ser177 has been demonstrated (20). In hPRL the corresponding serine is at position 179. Results using S179D, an analog of phosphorylated hPRL, are controversial since it is unclear if this modification activates or inhibits hPRL activity (21,22). In bovine PRL, Ser 90 is also phosphorylated (23) and this inhibits bPRL activity (24,25). Ser 90 is also conserved in hPRL but its phosphorylation remains to be determined. N- linked glycosylation at Asn 31 (26) was observed in hPRL and is believed to down-regulate PRL activity (27). Deamidation of hPRL (removal of ammonia from asparagine and glutamine) does not seem to affect its activity (28).

Several cleaved forms of hPRL have been identified. A 16 kDa fragment

(hPRL1-148) has been demonstrated in human pituitaries and sera (29). This

16 kDa fragment inhibits capillary endothelial cell proliferation (30), preventing

5 angiogenesis (31,32). The rat 16kDa PRL variant shows weak binding to the prolactin receptor (33). It is unclear at this time whether the 16 kDa fragment is formed as a result of alternative splicing of the RNA transcript or proteolytic cleavage of the full length hormone. Cleavage of hPRL is also attributed to the location of glycosylation sites near the cleavage sites (34). Finally, hPRL has been isolated in complexes with various molecules. Human prolactin

(glycosylated and un-glycosylated) was found in a complex with immunoglobulins (35,36). Un-glycosylated prolactin bound to immunoglobulin induces the growth of peripheral blood lymphocytes (37). A prolactin binding protein has been identified (similar to growth hormone) and is homologous to the extracellular domain of the prolactin receptor (hPRLbp) (38). A fraction of plasma prolactin is believed to circulate bound to hPRLbp.

Prolactin secretion was first discovered in acidophilic cells in the anterior pituitary called lactotrophes and hence was thought to be an exclusively pituitary phenomenon (39). In 1991, Nagy and Berczi used hypophysectomized rats to show that other cell types also secrete prolactin and that the synthesis of extra-pituitary prolactin increased to compensate for the lack of pituitary prolactin (40). To date, extra-pituitary hPRL secretion has been demonstrated in the human decidua (during pregnancy), the myometrium, mammary epithelial cells, various immune cells (highest levels in thymocytes and natural killer cells), various parts of the brain, and in the skin

(16).

6 As would be expected from the wide distribution of its expression, prolactin has been implicated in many diverse functions (reviewed in(17)).

Mice and rats have been used as a model to study the role of prolactin in mammals. The primary role for prolactin is in reproduction. PRL stimulates mammary growth, production of milk proteins, induces progesterone production, inhibits estrogen production and in general regulates the estrus cycle, pregnancy and lactation. PRL+/- mice show normal mating and pregnancy. Mammary gland development is impaired and maternal behavior is reduced. In PRL-/- mice the females are sterile and show significant impairment in mammary gland development and maternal behavior is absent.

PRL-/- males demonstrate delayed fertility (17).

PRL is believed to play a role in immune system development and function. Among other functions, prolactin is believed to stimulate antibody production, inhibit apoptosis of lymphocytes, activate macrophages and produce superoxide anions to kill pathogens (reviewed in (16)). Most disease states are associated with changes in prolactin expression rather than mutations in the prolactin gene. Hyperprolactinemia is associated with several conditions affecting reproduction. Elevated PRL levels are found in various autoimmune disorders such as systemic lupus erythematosus and rheumatoid arthritis. Elevated levels are also observed in cystic fibrosis (16). Prolactin levels serve as a marker for organ rejection (16). Colon cancer (41,42),

7 activation of malignant B cells (37), lymphomas (43) and the proliferation of promyelocytes (44) have all been attributed to hPRL and receptor levels.

An intense area of focus is the involvement of PRL in breast cancer. In

1995, a study demonstrated that human breast epithelial cells express PRL and the prolactin receptor (45). Studies using human breast adenocarcinoma cell lines showed that they synthesized and secreted prolactin (46). Cells from human breast carcinomas expressed higher levels of the prolactin receptor as compared to adjacent normal tissue (47). An epidemiological study done on postmenopausal women showed elevated plasma prolactin levels and an increased risk for breast cancer (48). Another study has shown that the

BRCA1 susceptibility gene is upregulated in a PRL-dependent manner (49).

Human breast cancer cell lines treated with PRL show increased expression of focal adhesion kinase and paxillin, associated with increased motility in cells (a characteristic feature of carcinoma progression in vivo) (50). This has led to emphasis on developing prolactin antagonists as an approach to treat breast cancer.

1.4 Structure-function studies on prolactin

Human prolactin is a single chain polypeptide with a molecular weight of approximately 23 kDa. Human prolactin (hPRL), growth hormone (hGH) and placental lactogen (hPL) constitute a family of lactogenic hormones. All three hormones bind to and activate the prolactin (or lactogenic) receptor

8 (hPRLr) through which they induce their reproductive effects (reviewed in (51).

In addition, hGH also activates the growth hormone (or somatotrophic) receptor (hGHr), through which it induces its growth promoting effects. hGH and hPL share 85% amino acid homology. hGH and hPRL only share about

23% homology. Although there are no published crystal structures available for human prolactin, there are crystal structures available for human growth hormone and ovine placental lactogen, other members of the lactogenic hormone family (52-54). Based on these related molecules, hPRL is predicted to be a four-helix bundle protein with an up-up-down-down configuration.

There are six cysteine residues that form 3 disulfide bonds. This results in the formation of two small loops at the N- and C- terminus and one large central loop. The disulfide bonds are formed between Cys4 - Cys11, Cys58 - Cys174, and Cys191 - Cys199. Studies done on ovine PRL shows that reduction of the central disulfide bond by DTT significantly alters the structure of the protein and also abolished biological activity (55). Mutagenesis of Cys58 in hPRL abolishes biological activity and receptor binding (10). Mutation of this cysteine breaks the large disulfide loop and this probably disrupts the structure of the hormone.

Swiss Modeler (56-58) was used to generate a model for the 3- dimensional structure of hPRL (Figure 1.1). In our model, the first 13 residues are unstructured. Secondary structure is predominantly α helical with the rest of the molecule forming unstructured loops that connect the helices. Helix 1

9 starts at residue 14 and extends upwards to residue 42. Residues 43-75 form a long non-helical segment called mini-helix 1 that links helix 1 and helix 2.

Helix 2 is composed of residues 76-102 which is connected to helix 3 by a short loop of 7 residues. Helix three includes residues 111-136 and is linked to helix 4 by another long loop from residues 137-160. Helix 4 includes residues 161-193. The last 6 residues also appear to be unstructured.

There are several crystal structures available for hGH both in the free and receptor bound form (54,59-64). In addition, homology-scanning mutagenesis and alanine-scanning mutagenesis have identified three important regions in hGH important for receptor binding – the C-terminal end of helix 4, the loop region between helix 1 and 2 including residues 54 to74, and the N-terminal end of helix 1(65,66). Later on, these were identified as the regions needed for the first receptor binding (Site 1). Gel filtration and fluorescence quenching experiments coupled with mutagenesis identified the second receptor binding site (Site 2) between helices 1 and 3 (67). Gel filtration experiments also demonstrated that the receptor binding was sequential, with the first receptor binding at Site 1 of hGH followed by the second receptor binding at Site 2 (67). The work described above studied the mechanism of hGH binding to the somatotrophic receptor. Studies on hGH binding to the prolactin receptor also demonstrate sequential binding (68).

Helix 1

Helix 2

Site 1 Site 2 Helix 4 Helix 3

Figure 1.1 Model of human prolactin Ribbon diagram of a model of human prolactin generated using Swiss Modeler (http://www.expasy.ch/swissmod/SWISS-MODEL.html) based on homology to human growth hormone.

11 In the absence of structures, site directed mutagenesis has been used to identify important residues in the receptor binding surfaces of PRL. Some of the earliest mutagenesis work was carried out in bovine prolactin. Deletion of single amino acids from the center of each of the four helices in bovine PRL

(as judged from the structure of ovine GH) resulted in a loss of lactogenic activity (69). Substitution of Tyr 28 did not significantly alter biological activity but deletion of Tyr 28 completely abolished bovine PRL activity. This is believed to be due to a loss of amphipathic helix structure. Another study mutated selected residues in helix 1 and helix 4 and showed a decrease in mitogenic activity of bovine PRL (70). Mutagenesis of residues in helix 1 and helix 4 of hPRL resulted in decreased receptor binding and biological activity

(71). Helix 1 residues have a small effect on receptor binding but the major contributions seem to come from residues in helix 4. Key residues in helix 4 include K181, H180, and R176. Alanine scanning mutagenesis of residues

58-74 of human prolactin demonstrated the importance of this region for hPRL activity (10). C58, K69 and P66 were found to be critical for hPRL biological activity. Addition of 9 residues to the C terminus of hPRL decreased its biological activity (72). It is believed that these 9 residues will fold into the groove between helix 1 and 4. All the above data suggest that one of the receptor binding sites in hPRL involves residues in helix 1, helix 4 and the loop region between helix 1 and helix 2.

12 As mentioned earlier, the 16kDa variant of hPRL has very little lactogenic activity (33). This could be because it lacks the putative helix 4 and hence critical residues for receptor binding.

Attempts have been made to identify the second binding site on hPRL.

Alanine substitutions of residues on helix 1 and helix 3 did not affect the activity. However replacement with bulkier groups such as arginine and tryptophan significantly reduced biological activity (9). These groups are believed to sterically hinder receptor binding. The receptor binding sites in hPRL and hGH lie in similar regions of the hormone; however the positions are not identical. All the residues in hPRL that have so far been mutated and tested for effects on receptor binding or biological activity have been tabulated

(Table 1.1) and the location of some key residues shown in Figure 1.2.

Although some of the critical residues for receptor binding have been identified, the details of the mechanism of receptor binding have not been studied. Some work on the binding kinetics of PRL from various species to rabbit, rat and bovine prolactin receptors was done using surface plasmon resonance technology (73). These studies showed that the affinities of both the receptor binding sites were similar when the hormone and receptor were from homologous species. However, when hormone and receptor from heterologous species were tested, the affinity at Site 1 was much stronger than the affinity at Site 2. This aspect will be discussed in greater detail in

Chapter 3.

Residues Biological Activity* Binding Activity** Reference

R16A no change no change (71)

A22W ------(9)

V23A ------(71)

V24A no change ND (9)

L25A no change ND “

L25R -- --- “

L25W -- --- “

+: The biological activity/binding increased between 10-30% relative to wild type ++: The biological activity/binding increased between 30-70% relative to wild type +++: The biological activity/binding increased more than 70% relative to wild type -: The biological activity/binding decreased between 10-30% relative to wild type --: The biological activity/binding decreased between 30-70% relative to wild type ---: The biological activity/binding decreased more than 70% relative to wild type ND: not determined * Biological activity was determined in a lactogenic bioassay, using Nb2 rat lymphoma cells ** Binding affinity is usually determined using a 125I-hPRL binding assay, where increasing concentrations of unlabeled mutant hormones compete with labeled wild type PRL for binding to homogenates of cells expressing the prolactin receptor.

(continued)

Table 1.1 Amino acids mutated in human prolactin and the effects on biological activity and receptor binding

14 Table 1.1 (continued)

Residues Biological Activity* Binding Activity** Reference

S26A -- -- (9)

S26R ------“

S26W ------“

I29A -- ND “

H30A -- -- (71)

L32A - ND (9)

S34A no change -- (71)

F37A -- --- “

S38A no change -- (71)

C58A --- undetectable (10)

H59A -- - “

T60A -- no change “

S61A + + “

L63A -- - “

P66A -- -- “

E67A no change no change “

K69A ------“

E70A - + “

(continued) 15 Table 1.1 (continued)

Residues Biological Activity* Binding Activity** Reference

Q74A +++ + (10)

E110A ++ ND (9)

I112A ++ ND “

S114A ++ ND “

K115A + ND “

E118A + ND “

Q122A + ND “

R125A - ND “

E126A + ND “

G129R ------“

Y169A ------(71)

H173A ------(71)

R176A ------“

H180A ------“

K181A ------“

K181E ------“

N184A - - “

Y185A ------“

L188A -- -- “

R192A + + “ 16 Site 1 Site 2

Figure 1.2 Location of key residues in the putative Site 1 and Site 2 A22, V23, L25, and S26 lie in Helix 1 and are colored red. K69 lies in the loop between helix 1 and 2 and is colored cyan. G129R lies in helix 3 and is colored dark blue. The putative Site 1 and Site 2 are indicated.

17 1.5 The prolactin receptor

The prolactin receptor was first identified in the rabbit mammary gland in 1974 (74). The human prolactin receptor was first cloned from cDNA libraries of liver and breast cancer cells (75). The prolactin receptor gene is located on chromosome 5 and until recently was believed to consist of 10 exons (76). Eight of these exons (3-10) were believed to encode the protein sequence of the prolactin receptor. Exon 2 was part of the 5’-untranslated region. Several different first exons have been identified: hE13, hE1N and hE1N2-5 (76,77). Each forms the 5’ end of the UTR and the type of first exon transcribed depends on the promoter used. Irrespective of which exon 1 is transcribed, all translated proteins have the same sequence.

Between 1988 and 1991, three isoforms for the rat prolactin receptor were identified (78-80). It has long been believed that multiple isoforms exist in human tissues. However, only one isoform had been identified in humans until 1999 when the intermediate form of the human prolactin receptor was detected in a breast cancer cell line (81). The intermediate isoform is produced as a result of a RNA splicing event at a consensus splice site between residues 1009 and 1582 of exon 10. The deletion of 573 nucleotides results in a frameshift that introduced a stop codon 13 residues after the splice site. This results in a receptor isoform with a truncated intracellular cytoplasmic domain. While the intermediate isoform does not induce cell proliferation, expression of this isoform does enhance cell survival, suggesting

18 activation of some anti-apoptotic molecules. In addition, the intermediate form does not appear to activate some of the downstream signaling pathways that the long form activates.

In 2001, Maria Dufau’s group at the NIH identified an additional exon 11

(82). They demonstrated that alternative splicing between exon 10 and 11 generated two novel short forms of the prolactin receptor (S1a and S1b). Both these short forms had truncated intracellular domains, showed similar affinities for prolactin, and acted as dominant negative inhibitors of signaling by the long form of the receptor. More recently a novel receptor isoform with only one extracellular domain has been identified (83). The N-terminal domain is deleted but the receptor can still bind to hPRL (although with lower affinity).

The prolactin receptor belongs to a family of class 1 cytokine receptors which includes the growth hormone receptor, receptors of several interleukins, leukemia inhibitory factor, granulocyte macrophage –colony stimulating factor

(GM-CSF), Oncostatin M, thrombopoetin and leptin, among others (17). All these receptors are classified together based on highly conserved sequences in both the intracellular and extracellular domains.

The receptor can be divided into three domains – the extracellular domain, the intracellular domain and a single pass transmembrane domain.

There are no crystal structures for the intracellular domain of the prolactin receptor. However there is a crystal structure of hGH bound to the extracellular domain of the hPRLr (59)(Figure 1.3). The extracellular domain

19 of the human prolactin receptor (hPRLrECD) is composed of 210 amino acids.

The ECD is divided into two domains – D1 and D2 (17). Both domains are analogous to the fibronectin type III module and are arranged in seven antiparallel β-strands. D1 is believed to interact with PRL and D2 is involved in interactions with the D2 domain of the second receptor. The two highly conserved features of the ECD and the family of receptors are the presence of two disulfide bonds in the N-terminal D1 and a “WSXWS” motif in the membrane proximal region of D2. Mutations of the cysteines cause structural changes in the receptor that affect its function (84). Mutations in the WSXWS motif affect ligand affinity although this motif lies distal to the ligand binding site (85). It is thought that this motif might affect the correct folding and trafficking of the receptor (86). Two tryptophan residues at positions 72 and

139 are important for ligand binding (86). There are three asparagine-linked glycosylation sites but their involvement is primarily in cell surface targeting

(59,87). The transmembrane domain is 24 amino acids long and highly hydrophobic. The intracellular region contains two highly conserved regions called Box 1 and Box 2. Box 1 is close to the membrane and contains a stretch of eight proline and hydrophobic residues (residues 243-250) -

IFPPVPGP-. Box 2 contains a succession of hydrophobic, negatively charged and positively charged residues (17).

20 Several studies showed that the prolactin receptor undergoes dimerization upon ligand binding. The initial studies showed that bivalent and not monovalent antibodies to the receptor could produce downstream effects of receptor activation (88). Other studies used ovine PL and GH to demonstrate receptor dimerization (89,90). The best piece of evidence was obtained using surface plasmon resonance technology. Studies demonstrated a 1:2 stoichiometry of hormone and receptor using the rat, bovine and rabbit prolactin receptors (73).

Receptor dimerization activates downstream signaling pathways, primarily the JAK/STAT pathway (17). JAK1 and JAK2 mediate prolactin receptor signaling (91-93) but it appears that JAK2 is the critical signaling molecule (94). JAK 2 activation involves autophosphorylation which occurs in less than a minute after ligand binding (91-93). The proline at 250 (part of the proline and hydrophobic residue rich region) and the conserved Box 2 region are critical for activation of the signaling cascade (95,96) Unlike the growth hormone receptor, JAK2 is constitutively associated with the prolactin receptor

(93,97). JAK2 phosphorylation and activation recruit STAT1, STAT3 and

STAT5, members of the STAT family of transcription factors. STAT5 appears to be the key factor. JAK2 mediated phosphorylation of STAT5 induces

STAT5 dimerization and translocation to the nucleus where gene transcription is initiated. The signaling cascade has been well reviewed in (17) and (18).

N-terminal domain (D1)

C-terminal domain (D2)

Figure 1.3 Extracellular domain of the prolactin receptor Ribbon diagram of the extracellular domain of the hPRLbp (from amino acids 1-211). PDB # 1BP3 (59)

22 Besides the JAK/STAT pathway, Src and Tec tyrosine kinases, and Vav guanine nucleotide exchange factor are also recruited by the prolactin receptor

(98,99). In addition the MAP Kinase pathway, the PI3 Kinase pathway,

Protein kinase C and others have been implicated (reviewed in (18)). The prolactin-lactogenic receptor complex is internalized into vesicles within 30 minutes of ligand binding (100,101). These vesicles are then degraded by the lysosome (17,102).

Based on this review of the literature, it is clear that unlike growth hormone receptor binding, the details of prolactin receptor binding are not well defined. Although prolactin receptor binding is believed to be sequential, no studies have clearly demonstrated that. Also, the kinetics of hPRL binding to its receptor has not been studied. Most of the work has focused on identifying residues in the putative binding pockets important for receptor interactions.

No studies have been done on residues outside of the binding pockets to determine how they articulate and allow for receptor binding. The work in this dissertation attempts to answer some of these questions.

Since hPRL is found in plasma bound to a binding protein analogous to the extracellular domain of the prolactin receptor (hPRLbp) and working with the full-length receptor is very difficult (due to aggregation), most of the receptor binding studies in vitro are done using the extracellular domain of the receptor. This is also the case for the work presented here and the receptor will henceforth be referred to as hPRLbp.

CHAPTER 2

MATERIALS AND METHODS

The materials and methods used in the rest of the dissertation will be described here. Most of the procedures described here are standard protocols commonly used in the laboratory.

2.1 The pT7-7 phagemid

We received the pT7-7 expression plasmid from S. Tabor (Harvard

Medical School, Boston, MA). The vector includes a T7 RNA polymerase driven promoter that can be used in E. coli strains that express T7 RNA polymerase. Dr. Francis Peterson incorporated an f1 origin of replication into the pT7-7 plasmid, generating the pT7-7f(-) phagemid (103). This phagemid is used for site-directed mutagenesis and protein expression. The plasmid also has an ampicillin resistance marker that can be used for positive selection of transformants. Dr. Francis Peterson cloned the hPRL gene into the pT7-7 phagemid and Dr. Karen Duda cloned the hPRLbp gene into the phagemid.

2.2 Primer Design for Site Directed Mutagenesis

All the primers used were designed using similar logic. They were between 18-30 bases in length. The sequence to be mutated lay approximately at the middle of the sequence to ensure homology at the 5’ and

3’ ends for optimal annealing conditions. In addition the primer ends were GC rich to increase stability of the template-primer complex during annealing and extension. The primers were designed to include a unique translationally- silent restriction site as a tool to rapidly screen for positive clones. The Primer

Generator (http://www.med.jhu.edu/medcenter/primer/primer.cgi)- a public domain software, was used to design the silent restriction sites. The primers were ordered from Integrated DNA Technologies (Coralville, IA).

2.3 Site-directed mutagenesis

RZ1032 cells, an E coli strain lacking dUTPase and uracil N- glycosylase were transformed with the wild-type hPRL gene in the pT7-7 phagemid. Because of the deficient enzymes, this bacterial strain allows the incorporation of uracil into DNA. Single stranded DNA was synthesized by infecting the cells with the R408 helper phage (Promega, Madison, WI) at a multiplicity of infection (MOI) of 20. The single stranded DNA was purified using phenol-chloroform extraction and ethanol precipitation and used for site directed mutagenesis.

25 Site directed mutagenesis was performed using the Kunkel method

(104). This method was preferred over the PCR method because there is no sub-cloning involved and multiple mutations can be introduced at the same time. The single stranded DNA was incubated with phosphorylated primer containing the desired mutation. Second strand synthesis and ligation was facilitated by T7 RNA polymerase (New England Biolabs, Beverly, MA) and T4

DNA ligase. The double stranded DNA was then transformed into DH5α cells.

The colonies were screened using the translationally-silent restriction sites introduced in the mutagenesis primer. Positive clones were confirmed by

Sanger dideoxy sequencing (105) at the Neurobiotechnology Center

Sequencing Facility, The Ohio State University, using the ABI 373XL Stretch

DNA Sequencer.

2.4 Expression, folding and purification of proteins

Wild-type prolactin, the various prolactin mutants and the prolactin receptor were expressed in BL21 (DE3) cells as previously described (103).

Briefly, the respective phagemids were transformed into BL21(DE3) cells

(Novagen, Madison, WI). A single colony was selected and grown in 8 ml of

LB broth for 6-8 hours. This culture was then inoculated into 1 liter of LB broth and grown to an OD600 of 0.3-0.4. Protein expression was induced using 0.4 mM isopropyl thiogalactoside (Sigma, St. Louis, MO). After 3-4 hours, the cells were collected by centrifugation and lysed using a French Pressure cell

26 (SLM-Aminco, Urbana, IL). The inclusion bodies were collected by high-speed centrifugation and re-suspended in a 4.5 M urea solution at an alkaline pH.

For proteins with even numbers of cysteine residues, the inclusion bodies were re-suspended in 100 ml of the urea solution while the proteins with an odd numbers of cysteine residues were re-suspended in 400 ml of 4.5 M urea.

This was done to dilute the proteins during the refolding process to minimize cysteine-based dimer formation. The proteins were refolded by extensive dialysis against 20 mM Tris, pH 7.5. Proteins with an odd number of cysteines had 2-mercaptoethanol and hydroxyl ethyl disulfide added during the first two dialysis steps to prevent disulfide bond formation till the proteins were correctly refolded. The proteins were purified by anion-exchange chromatography using a DEAE Sepharose Fast Flow column (Amersham Biosciences,

Piscataway, NJ) with an ÄKTA explorer chromatograph (Amersham

Biosciences, Piscataway, NJ). A salt gradient from 0-500 mM NaCl was used to elute the protein. Peaks with 280:250 nm absorption ratios of 2 or higher were collected. Prolactin has a characteristic 280:250 nm ratio of 2 and the receptor has a ratio of 2.2. The purified proteins were dialyzed against 10 mM ammonium bicarbonate, lyophilized and stored at -30°C.

27 2.5 Characterization of recombinant proteins

2.5.1SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Between 15 and 20µg of each protein was run on a 15% SDS-PAGE gel under reducing and non-reducing conditions. A reducing gel (with 2- mercaptoethanol) was used to determine the size and purity of the recombinant protein preparations. A non-reducing gel was used to verify correct disulfide bond formation by comparing the hydrodynamic radius of the mutants to that of the wild-type hormone and the lack of disulfide-based dimers. Incorrect disulfide bond formation would alter the hydrodynamic radius of the protein and is detected as a difference in apparent molecular weight. The recombinant wild-type hormone has previously been compared to a preparation from a biological source. The gel images were digitally recorded using an Alphaimager 2000 (Alpha Innotech Corporation, San Leandro, CA).

2.5.2 UV Absorption Spectroscopy

UV absorption spectra between 200-350 nm are useful for three pieces of information. At 350 nm, proteins do not absorb light but protein aggregates scatter light, so the intensity of absorption reflects the quality of the protein preparation. The aromatic amino acids, Trp, Tyr and Phe absorb at peaks of

280 nm, 274nm and 257nm respectively. The peak height is reflective of the protein concentration and the wavelength of maximal absorption is a reflection of the hydration of the aromatic amino acids. The disulfide bonds partly

28 contribute to the absorption at 250 nm. Any change in the disulfide bond angle or length changes the absorption at 250 nm. Protein solutions at 25 µM concentration were prepared in 20 mM Tris, pH 8.2, 150 mM NaCl.

Absorption spectra from 200-350 nm for all the proteins were collected at 20°C on a Lambda 45 UV/VIS Spectrometer (Perkin Elmer, Wellesley, MA).

2.5.3 Fluorescence Spectroscopy

The three aromatic amino acids, tryptophan, tyrosine and phenylalanine also fluoresce. The maximum absorption wavelength is 280nm, 274nm and

257nm respectively. The emission spectrum of Trp lies between 307 to 353 nm and is influenced by the extent of exposure to water. The emission maximum for Tyr and Phe are not significantly affected by their extent of hydration. Each protein has a characteristic emission spectrum and the various hPRL mutants were compared to the wild-type to ensure that the mutation did not affect the emission spectrum. A shift in the wavelength of maximal emission of more than 2nm was considered significant. The peak height is a measure of the number of Trp residues in the solution and is a reflection of the protein concentration. Proteins at 1 µM concentrations in 10 mM Tris pH 8.2, 150 mM NaCl were excited at 285 nm to minimize contributions from Phe and Tyr. The emission spectrum was followed from

300 nm to 400 nm on a LS 45 Luminescence Spectrometer (Perkin Elmer,

Wellesley, MA).

29 2.5.4 Circular Dichroism (CD) Spectroscopy

CD spectroscopy measures the secondary structure of proteins. α- helices, β-sheets and random coiled proteins have a characteristic CD spectrum resulting from the properties of the amide groups of the peptide backbone. α-helices show two characteristic negative peaks at 222 nm and

208 nm, and a positive peak at 190 nm. β-sheets show a characteristic negative peak at around 218 nm. Perturbations in the secondary structure of proteins will cause a change in the CD spectrum and this could alter the biological activity of the hormone. To ensure that the various mutants did not show a change in secondary structure, 25 µM concentrations of the proteins were prepared in 10 mM Tris pH 8.2, 150 mM NaCl and the far-UV CD spectrum from 200 nm to 260 nm was collected at 20°C using an AVIV

Circular Dichroism Spectrometer Model 202 (Aviv Instruments, Lakewood,

NJ). CD spectra of proteins are additive. We tested whether receptor binding induced a conformation change in the hormone by determining the CD spectrum of a mixture of the hormone and receptor and comparing it to the sum of the CD spectra of the free hormone and receptor.

2.5.5 Mass Spectrometry

The molecular weight of selected mutant proteins was confirmed using mass spectrometry. 20 µg of the proteins were cleaned using a peptide trap

(with a reverse-phase HPLC packing) (Michrome Bioresources Inc., Auburn, 30 CA) to remove salts or other contaminants. Proteins were eluted from the peptide trap using 50% acetonitrile and provided to the Campus Chemical

Instrumentation Center (CCIC), The Ohio State University, where the experiments were performed using a Micromass Q-TOF II mass spectrometer

(Milford, MA).

2.6 Lactogenic Biological assays

FDC-P1 cells containing the prolactin receptor were a gift from

Genentech Inc. (South San Francisco, CA). Cells were maintained in RPMI

1640 medium (GIBCO, Grand Island, NY) containing 10% fetal bovine serum

(Novagen, Madison, WI), 10 ng/ml recombinant human IL-3 (Peprotech Inc.,

Rocky Hill, NJ) and 220 µg/ml G418 sulfate (Invitrogen, Carlsbad, CA). Cells in log phase were washed three times in RPMI 1640 without phenol red

(Sigma, St Louis, MO) supplemented with 10% equine serum (Novagen,

Madison WI) and G418 sulfate (GIBCO, Grand Island, NY) and incubated in this medium for 24 hours prior to the assay. 15,000 cells were placed into each well of a 96-well plate. Concentrations of recombinant wild type and mutant hPRL were determined by the bichinchonic acid/ copper sulfate assays

(Pierce, Rockford, IL). The hormone was then diluted to the desired concentrations (0.01 nM to 10 µM) in RPMI 1640 medium without phenol red and added to triplicate wells of the 96-well plate. The final volume in each well was 100 µl. The plates were gently agitated to evenly spread the cells and

31 then incubated at 37°C in a humidified 5% CO2/95% air atmosphere for 48 hours. Hormone-induced proliferation was assessed using a vital dye (Alamar

Blue, Accumed International, West Lake, OH). The dose-response curve was used to determine the ED50 and ID50 values for the mutants using a four parameter fit method (106).

The activity of hPRLbp was determined in these cellular assays by measuring the ability of graded concentrations of hPRLbp to sequester hormone and reduce cell proliferation. 0.3 nM of hPRL was added to each of the wells along with increasing concentrations of hPRLbp from 0.01 nM to 10

µM. The rest of the assay was performed as described above.

2.7 Surface Plasmon Resonance Experiments

When light of a certain wavelength at a low angle of incidence propagates through a medium of higher refractive index and hits a surface of lower refractive index, there is total internal reflection of the light. However some electrical field intensity is leaked as an evanescent field wave into the medium of lower refractive index. If the interface between the two surfaces is coated with a conducting metal such as gold, the evanescent field wave excites surface plasmons in the metallic layer creating an enhanced evanescent wave. These waves can travel for a short distance through the medium of lower refractive index. This excitation of surface plasmons causes a decrease in the reflected light intensity at a given angle. When the refractive

32 index of the medium is changed, there is a change in the angle of decreased reflected light intensity. This change is detected by a two-dimensional detector array. This SPR technology can be used to study the time-dependent interaction between molecules. One of the molecules is attached to a dextran- coated gold surface. The second molecule is then passed over this chip surface. If there is an interaction between the two molecules, there is an increased concentration of molecules on the chip surface which causes a change in the refractive index of the medium and that in turn changes the angle of decreased reflected light intensity. This change in refractive index is dependent upon the rate and number of molecules bound to the surface and is a reflection of the rate of interaction between the two molecules. The optical bench (the source of incident light and the detector array) are located on the opposite side of the surface where the interactions take place. The data is presented as a change in the angle of decreased reflected light intensity over time and is measured in resonance units (RU).

2.7.1 Immobilization of hormones on the dextran-coated chip surface

The optical biosensor used was the BIAcore 3000 (Biacore Inc.,

Piscataway, NJ). All reagents were purchased from BIAcore Inc. The different mutant hormones were bound to the dextran surface of a CM5 sensor chip using ligand thiol coupling. This is a general purpose chip where a 50 nm thick gold surface is coated with carboxymethylated dextran matrix to which

33 proteins can be covalently attached. The flow rate was set at 5 µl/min. The buffer used was 10 mM Hepes, 150 mM NaCl, 3 mM EDTA, 0.005%

Surfactant P20 (HBS-EP buffer). The chip surface was activated by injecting

10 µl of a mixture of 0.05 M N-hydroxysuccinimide/ 0.02 M N-ethyl-N’-

(dimethylaminopropyl)-carbodiimide (NHS/EDC). Disulfide groups were introduced on the activated chip surface using a 20 µl injection of 80 mM 2-(2- pyridinyldithio) ethaneamine (PDEA) in 0.1 M sodium borate buffer pH 8.5.

The hormones at 200 nM concentrations in 10 mM sodium acetate buffer pH

4.3 were coupled to the chip surface such that roughly 100 RU of hormone was bound, corresponding to about 0.1 ng/mm2 ligand concentration. The unreacted disulfide groups on the chip surface were blocked using a 20 µl injection of 50 mM cysteine / 1 M NaCl in 0.1 M sodium formate buffer pH 4.3.

Before the various mutants were bound to the CM5 sensor chip, they were treated with DTT to reduce the free cysteine. The proteins in solution were incubated with a 5 µM excess of DTT for 5 minutes at room temperature.

The DTT was then separated using a Microcon YM-10 centrifugal concentrator

(Millipore Corporation, Bedford, MA). The centrifugation was repeated twice to ensure that over 95% of the DTT was removed. The reduced proteins were then compared to un-treated proteins by UV spectroscopy to ensure that the

DTT treatment did not affect disulfide bond formation.

34 2.7.2 Mass transport limitation

The interaction between the hormone and the receptor is limited by how quickly the receptor is transported from the solution to the chip surface and this is called mass transport. This can affect the observed association and dissociation constants. If the receptor is rapidly depleted from the solution the rate constants will be limited by the rate of mass transport. Two ways to minimize mass transport limitations is to increase the flow rate and to optimize the ligand concentration on the chip surface.

Mass transport limitations were tested by flowing 50 nM receptor across a chip with 100RU of hormone bound at 5 µl/min, 15 µl/min and 75 µl/min.

The initial rates of binding were calculated. A variation of more than 10% in the rates is a sign that mass transport may influence the kinetic data.

2.7.3 Equilibrium binding experiments

M158C, G129C and K181C were coupled to the CM5 sensor chip at various densities ranging from 100 RU to 1000 RU. 100 µM of hPRLbp was flowed over the chip surface at 50 µl/min so as to saturate all the receptor binding sites on the coupled hormone irrespective of the receptor binding affinities at the two sites on the hormone. The RU of receptor bound per RU of hormone bound was calculated at 200 seconds using the formula

Receptor = RU of receptor X Mol. Wt. of hormone Hormone RU of hormone Mol. Wt. of receptor

35 2.7.3 Binding of hPRLbp to the hPRL mutants

Varying concentrations of the prolactin receptor from 50 nM to 1000 nM were passed in a random order at 50 µl/min over the chip surface for 5 minutes to follow the binding kinetics. The dissociation kinetics were followed for 5 minutes by passing HBS-EP buffer over the chip surface. Finally, the chip surface was regenerated to remove any remaining bound receptor using a 30 second injection of 2.5 M MgCl2. The binding curves were then subtracted from a blank surface which was activated and blocked with cysteine but did not have any hormone attached to it. The receptor binding curves were then subtracted from the curve for an injection of buffer alone without any protein.

2.7.4 Evaluation of the kinetic data

BIAevaluation 3.0 (BIAcore Inc., Piscataway, NJ) software was used to fit the kinetic data to models for 1:1 binding (for the mutant where Site 2 was blocked) and a two-site model for the mutant with both binding sites available.

The association and dissociation rate constants were determined and the equilibrium constants were calculated from these values. The 1:1 (Langmuir) binding model available with BIAevaluation 3.0 was used to fit the data for the

G129C mutant where only one binding site was available. For all the mutants where both binding sites were available, the following equations (developed by

Dr. Jeffrey Canfield) were used.

H00 + R H10R (1)

H10R + R H11RR (2)

The first receptor [R] binds to the free hormone [H00] at Site 1 forming a

1:1 hormone receptor complex [H10R]. The second receptor then binds to the hormone at Site 2 forming the heterotrimeric complex [H11RR]. ka1 and kd1 represent the association rate constant and dissociation rate constant for equation (1) and reflect binding at Site 1. ka2 and kd2 represent the association rate constant and dissociation rate constant for equation (2) and reflect binding at Site 2. KD1 and KD2 represent the equilibrium dissociation constants for Site

1 and Site 2 respectively. The data fitting is an iterative process beginning with the assignment of initial values for the reaction rate constants which are then optimized by minimizing the sum of the squared residuals. The initial

3 -1 -1 -5 -1 parameters for ka1 and ka2 were set at 10 M s , kd1 was set at 10 s and kd2 was set at 10-7 s-1.

2.8 Fluorescence Resonance Energy Transfer (FRET)

Wild-type hPRL and the cysteine mutants, M158C, G129C and K181C, were labeled with 7-diethylamino-3-(4’-maleimidylphenyl)-4-methylcoumarin

(CPM) (Molecular Probes, Eugene, OR). The hormones at 100 µM concentration were mixed with a five-fold molar excess of CPM and incubated

37 at room temperature with shaking for 4 hours. The mixture was then passed through a Sephadex G50 column (Amersham Biosciences, Piscataway, NJ) to separate the labeled hormone from the free fluorochrome. The labeling of the hormone was verified using the Micromass Q-TOF II mass spectrometer at the

CCIC, The Ohio State University.

1 µM of the labeled hormones was mixed with various concentration of the receptor as indicated. For the competition experiments, 1 µM of the labeled hormone was mixed with 1 µM of the receptor and 50 µM of unlabeled wild-type prolactin. The samples were allowed to reach equilibrium at room temperature for 1 hour. Fluorescence spectra were collected using 295 nm as the excitation wavelength and monitoring the emission spectra from 300 to

575nm.

CHAPTER 3

MECHANISM OF ORDERED LACTOGEN RECEPTOR BINDING

3.1 Introduction

Binding of the receptors to hPRL is believed to be sequential with the first receptor binding at Site 1 followed by the second receptor binding at Site

2 (9,10). However, the mechanism of sequential binding is unclear. The current theory is that there is a differential affinity for the receptor at the two binding sites (73). Site 1 has a greater affinity for the receptor and hence the receptor binds to Site 1 first, followed by the second receptor binding at Site 2.

Evidence from other sources as well as the results presented here support an induced-fite model for PRL receptor binding rather that the differential affinity model. Examination of the available crystal structures of the related hGH suggests that there is a conformation change in hGH after the first receptor binds to the hormone (52,59). Specifically, there are three changes observed.

There is an extension of helix 2 into the loop connecting helix 2 and 3. There is also a restructuring of mini-helix 1 causing an increase in helical content.

K181 Site2 Site2 Site1 Site1 G129

M158

Site 2 Site 1

Figure 3.1 Location of residues in hPRL mutated to cysteine K181 lies in Site 1, G129 lies in Site 2 and M158 lies distal to the two binding sites

40 Finally, there is a rotation of helix 1 relative to helix 3 by about 15°. The second receptor-binding site lies between helix 1 and helix 3. We believe that in the free state, the first binding site is available for receptor binding.

However the second receptor binding surface is disorganized. When the first receptor binds to the hormone, there is a spatial reorganization of the residues that constitute the second receptor binding surface creating binding site.

To test this hypothesis, residues previously identified as important within either Site 1 or Site 2 (Table 1.1) were mutated to cysteine. At Site 1 we mutated Lys 181 (71) and in Site 2 we mutated Gly 129 (9). As a control, we mutated Met 158, which is located distal to the two binding surfaces at the top of helix 4. This residue was selected based on the model of hPRL (Figure

1.1). The location of these residues is shown in figure 3.1. Based on the model, these residues extend out into the region between the two helices making them easily available for manipulation. We then coupled these cysteine mutants to a CM5 dextran coated sensor chip from BIAcore via a disulfide bond. The dextran on the chip surface provides a steric block at the respective binding site. Receptor binding can then be determined by flowing receptor across the chip with the various ligands coupled to it. Equilibrium experiments were performed to measure the relative amounts of receptor associated with each constraint. Kinetic experiments were also performed to determine whether the affinities at the two binding sites were similar or different (either supporting or contradicting our theory).

41 3.2 Results

3.2.1 Characterization of the Recombinant Proteins

Five to ten batches of each of the mutant proteins were prepared and the yield varied between 20-50 mg per liter. The purified recombinant proteins were run on both reducing and non-reducing SDS-PAGE gels (Figure 3.2).

Reducing gels showed a single band between 20-24 kDa in molecular weight for wild-type hPRL and the hPRL mutants. All the recombinant protein preparations thus appear to be over 95% pure. Non-reducing gels provide information about disulfide bond-formation in the proteins (as a reflection of the hydrodynamic radius). All the hPRL proteins appear to run at the same relative position on the gel between 20-24 kDa. This suggests that disulfide bond formation was not affected by the presence of an additional cysteine.

Traces of dimer were present in preparations of M158C hPRL, G129C hPRL,

I146C hPRL and hPRLbp. However the proportion of dimer is very small compared to the monomeric population. The molecular weight markers were run under reducing conditions even on the non-reducing gel. The reducing gel of hPRLbp showed a single band with a molecular weight around 30 kDa.

This is a little higher than predicted but there is no information available in the literature and there is no standard to compare it to. DNA sequencing suggests that the gene sequence is accurate so it is possible that the protein just runs at a higher molecular weight. The non-reducing gel of hPRLbp also shows trace amounts of dimer but the proportion of dimer to monomer is very small.

42 The proteins were then characterized by various spectroscopic methods. UV spectroscopic analysis of the hPRL cysteine mutants showed that they all had an absorption peak around 277 nm, characteristic for the aromatic amino acids. All the proteins also had a 280/250 ratio between 1.8 and 2. Thus, disulfide bond formation was not affected by introduction of the seventh cysteine. No light scattering was observed at 350 nm indicating that none of the proteins are aggregates (Figure 3.3A). Fluorescence spectra of the cysteine mutants compared to the wild-type showed that introducing the seventh cysteine into hPRL did not shift the peak maxima from 340 nm. Thus the environment surrounding the tryptophan residues is not altered by the free cysteine (Figure 3.3B). The variations in the peak height of the fluorescence spectra suggest that there are more tryptophan residues in solution and could result from variations in protein concentrations. CD spectra of the cysteine mutants compared to wild-type hPRL showed the characteristic curves observed for proteins that are predominantly α-helical (Figure 3.3C). There were two negative peaks at 222 nm and 208 nm and a positive peak approaching 190 nm. The CD spectra for all the proteins overlay very well

(Figure 3.3C inset), indicating that the free cysteine did not alter the environment around the helices. Overall, no conformation changes were detected in the cysteine mutants M158C, G129C and K181C as compared to the wild-type hPRL.

43 The receptor was also characterized by the same spectroscopic methods. The maximum absorption in the UV spectrum was at 280 nm

(Figure 3.4 A) and the 280/250 ratio was 2.0. Fluorescence emission spectra showed an emission maximum at 345 nm (Figure 3.4 B) and the CD spectrum was characteristic of proteins that are predominantly β-sheet with a negative peak at 215 nm Figure 3.4 C). Over ten different batches of hPRLbp were prepared with yields ranging from 20-50 mg per liter of cells. The preparations were found to be similar to receptor batches previously expressed in the laboratory.

The hPRL mutants were then tested in lactogenic bioassays to compare changes in their biological activity relative to the wild-type (Figure

3.5). We observed that the M158C mutant had similar activity to the wild-type with both proteins having ED50 values around 0.8nM. Both G129C and K181C showed a decrease in peak height along with a right shift in the agonist phase of the curve. The ED50 values were 3.08nM and 37.83nM respectively. The changes in ED50 values are not unusual since the mutations lie in the receptor binding pockets and could sterically hinder receptor binding. The recombinant hPRLbp preparation was tested in lactogenic bioassays. As receptor concentration increased, there was a decrease in cellular activity suggesting that hPRLbp could successfully compete with the receptors on the cell surface for binding hPRL, demonstrating that the receptor preparation was biologically active (Figure 3.6).

44 Initial attempts to couple the hormone to the CM5 sensor chip failed.

Mass spectrometry indicated that the molecular weight of the proteins with a free cysteine was about 78 Daltons heavier that the predicted molecular weight. 2-mercaptoethanol (2-ME) is used during the initial stages of the refolding process of proteins with an odd number of cysteine residues to allow the proteins to fold correctly before disulfide bond formation. 2-ME has a molecular weight of 78 Daltons and might be linked to the free cysteine residues in the mutants. In order to couple the hormone to the CM5 chip, the free cysteine was reduced with DTT. 5 µM molar excess of DTT was added to the protein samples and incubated for 5 minutes at room temperature. At these concentrations, it is unlikely that any stable disulfide bonds will be reduced. The DTT was then separated from the proteins using a centrifugal concentrator as described in the materials and methods. Since there was the possibility that the DTT might break the internal disulfide bonds, UV spectroscopy of the reduced proteins was carried out to ensure that disulfide bond formation was not affected (Figure 3.7). Both the reduced and untreated proteins showed a characteristic absorption maximum at around 278 nm and a

280/250 ratio of 1.9-2.1. There was no light scatter observed at 350 nm with any of the proteins. This indicates that the three internal disulfide bonds in the cysteine mutants were not altered by the treatment with DTT.

45 P rB L M M C M C C C R M T 8 6 M 9 1 P W W 5 W 4 W 2 8 h W W 1 1 1 1 M M M M M I M G K

66 66 66 66 66 45 45 45 45 45 36 36 36 36 36 29 29 29 29 29 24 24 24 24 24 20.1 20.1 20.1 20.1 20.1

14.2 14.2 14.2 14.2 14.2

Reducing Gel

P B C M M 8 M C C M L M 5 C W 9 1 R W T 1 W 6 2 8 W M W W M 4 M 1 1 P M M I1 G K M h

66 66 66 66 66 45 45 45 45 45 36 36 36 36 29 36 24 29 29 29 29 24 20.1 24 24 24

20.1 20.1 20.1 14.2 20.1

14.2 14.2 14.2 14.2

Non-Reducing Gel

Figure 3.2 SDS-PAGE gels of recombinant protein preparations Samples were prepared in the presence (top) or absence (bottom) of 2- mercaptoethanol and resolved on a 15% SDS-PAGE gel.

46 A

0.6

0.5

) 0.4 U

0.3 bance (A sor b

A 0.2

Wild Type hPRL 0.1 hPRL M158C hPRL G129C hPRL K181C 0.0 200 220 240 260 280 300 320 340 Wavelength (nm)

(Continued)

Figure 3.3 Spectroscopic Characterization of the Cysteine Mutants UV (A), Fluorescence (B) and CD (C) spectra of the binding site cysteine mutants. Samples were prepared at 25 µM (A and C) and 1 µM (B) concentrations in 10 mM Tris, pH 8.2, 150 mM NaCl. Spectra were collected at 20-25°C. Inset in each of the cases is data normalized to 277 nm (A), 340 nm (B), 222 nm (C).

(Figure 3.3 continued)

600 B ) 500 units y

itrar 400 b r a (

ity 300 ns Inte e

c 200 n e

res Wild Type hPRL 100 hPRL M158C

Fluo hPRL G129C hPRL K181C 0 300 320 340 360 380 400 Wavelength (nm) C

Wild Type hPRL hPRL M158C 0 hPRL G129C

-1 hPRL K181C l mo 2 m

g.c -5000 de ) θ (

y t i c i -10000 lar Ellipt o M

-15000

200 210 220 230 240 250 260 Wavelength (nm)

48 A

1.4 hPRLbp

1.2

1.0

0.8 rbance o s

b 0.6 A

0.4

0.2

0.0 220 240 260 280 300 320 340 Wavelength (nm) B 1.2

1.0 ity 0.8 tens n

e I 0.6 enc esc 0.4 Fluor

0.2

0.0 300 320 340 360 380 400 Wavelength (nm)

C -1 l mo 2 -1000 deg.cm (θ) ty

i -2000 liptic El

lar -3000 o M

-4000 200 210 220 230 240 250 260 Wavelength (nm)

Figure 3.4 UV (A), Fluorescence (B) and CD (C) spectroscopy of hPRLbp Samples were prepared at 25 µM (A and C) and 1 µM (B) concentrations in 10 mM Tris, pH 8.2, 150 mM NaCl. Spectra were collected at 20-25°C.

49 WThPRL 100 hPRL M158C hPRL G129C hPRL K181C 80 e

% Maximal Respons 20

0.01 0.1 1 10 100 1000 10000 Hormone Dose (nM)

Figure 3.5 Lactogenic bioassay of the binding site cysteine mutants Increasing hormone doses were incubated with FDC-P1 cells stable transfected with the prolactin receptor. Cell proliferation was assessed using a vital dye. The experiment was performed three times and representative data are presented here.

50 100 hPRLbp + 0.5nM hPRL

60 eduction 40 % R

0 0.01 0.1 1 10 100 1000 Receptor Dose (nM)

Figure 3.6 Competitive bioassay of hPRLbp. Increasing doses of hPRLbp were mixed with 0.5 nM hPRL and allowed to come to equilibrium. This mixture was added to FDC-P1 cells expressing the prolactin receptor. Cell proliferation was detected using a vital dye. The assay was performed on three different batches of hPRLbp and a representative assay is presented here.

1.4

1.2

1.0

sorbance 0.8 b ve A

i 0.6 Relat 0.4 WThPRL untreated M158C reduced G129C untreated 0.2 G129C reduced K181C untreated K181C reduced 0.0 220 240 260 280 300 320 340 Wavelength (nm)

Figure 3.7 UV spectroscopy comparing untreated proteins with samples treated with DTT 25 µM of the various proteins treated with 5 µM excess of DTT were prepared in 10 mM Tris, pH 8.2, 150 mM NaCl and absorption spectra collected from 220-350 nm at 25°C.

52 3.2.2 Effect of blocking Site 1 or Site 2 on receptor binding capacity

M158C, G129C and K181C hPRL mutants were coupled to a CM5 sensor chip at various densities from 100-1000 RU. 5 independent experiments were performed. A saturating concentration of the receptor (100

µM) was flowed across the chip surface and the amount of receptor binding determined (Figure 3.8). M158C shows the maximum binding capacity which is to be expected since both binding sites are available. G129C (with Site 2 blocked) had approximately 60% of the total amount of the receptor bound as compared to M158C while the Site 1 blocked mutant (k181C) lost about 95% of the binding capacity as compared to M158C (Figure 3.8 lower panel). This suggests that receptor binding at Site 2 is dependent on receptor occupancy at Site 1, but receptor binding at Site 1 is independent of receptor binding at

Site 2. If the theory that the two receptor binding sites had different affinities is correct, at such high receptor concentrations, Site 2 would most likely be saturated with receptor. The amount of receptor bound per RU of ligand was calculated as described in the experimental methods and is shown in the lower panel of Figure 3.8. The average RU of receptor bound per RU of ligand attached was calculated for M158C, G129C and K181C and was found to be

1.87 (±0.4), 1.25 (±0.4) and 0.09 (±0.08) respectively. There is some variation in the ratio of receptor bound per ligand attached to the chip. One possible explanation could be that the ligand is sometimes attached to the chip so that adjacent dextran chains sterically block the available receptor binding site.

53 A cysteine mutation was introduced in the loop between helix 3 and 4 to ensure that the observations with M158C are not due to the effect of the mutation. Ile146 was mutated to cysteine, and purified as described in the materials and methods. I146C and M158C were compared in SPR experiments. About 100 RU of both mutants were coupled to a CM5 sensor chip and 1 µM of hPRLbp was passed over the chip at 50 µl/min (Figure 3.9).

Both M158C and I146C bound approximately 50 RU of receptor. In addition, the binding curves of M158C and I146C overlay extremely well. Any significant changes in the association or dissociation rate constants would significantly alter the shapes of the curves. This suggests that the observations with M158C are not an artifact of the mutation but reflect the binding properties of wild-type hPRL.

3.2.3 Kinetics of receptor binding to M158C and G129C

The kinetic parameters for interactions between M158C or G129C and the receptor were determined using the different models described in the experimental procedures (Figure 3.10, Table 3.1). The experiment was performed a minimum of three times. Various concentrations of hPRLbp (50 nM, 80 nM, 100 nM, 200 nM, 500 nM, and 800 nM) were flowed at 50 µl/min in random order over a CM5 chip to which 100 RU of M158C, G129C or K181C were bound. The binding curves for M158C were fit to the two site sequential binding model (Figure 3.10 A) and the curves for G129C were fit to the 1:1

54 Langmuir binding model (Figure 3.10 B). The binding curves from K181C could not accurately be fit since the amount of receptor bound was too low

(less than 5 RU, Figure 3.10 C). Chi2 values for the fit were less than 5 indicating a good fit and the residuals (which are a measure of the variance between the predicted model and the experimental data) were less than 4 RU.

The experimental data for M158C and G129C fit the predicted model very well.

The association rate constants and dissociation rate constants for

M158C and G129C are presented in Table 3.1. The data are an average of at least three experiments. The equilibrium dissociation constant was derived from the average rate constants. The rate constants and derived equilibrium constants for Site 1 did not significantly change (p > 0.05) whether Site 2 was blocked or not (compare ka1, kd1 and KD1 for M158C and G129C, Table 3.1).

Thus, binding of the receptor to Site 1 was not affected by the presence or absence of a functional Site 2. In addition, the affinities at Site 1 and Site 2 appear to be very similar (109 nM for Site 1 compared to 153 nM for Site 2).

Based on the data presented above, it appears that the differential affinity model may not accurately describe the mechanism of hPRL – hPRLbp interactions.

55 RU 800

700

600

500 M158C 400

300 G129C 200

100

0 K181C -100

-200

-300 -100 -30 40 110 180 250 320 390 460 530 600 Time sec

2.5

2.0

1.5

1.0 Ratio[Analyte/Ligand]

0.5

0.0 M158C G129C K181C Mutant

Figure 3.8 Equilibrium binding of hPRLbp to the cysteine mutants 100 µM of hPRL bp was flowed at 50 µl/min over a CM5 sensor chip to which 250-300 RU of the cysteine mutants were bound. Receptor binding was followed for 4 minutes and then receptor dissocitation followed for another 4 minutes. The amount of receptor bound per RU of ligand on the chip is quantified in the lower panel. The data are an average of 5 experiments and error bars indicate standard deviation. 56

RU 100

R 40 e s pons 20 e M158C 0

-20 I146C

-40

-60

-80 -50 0 50 100 150 200 250 300 350 400 450 500

Time sec

Figure 3.9 Comparison of hPRLbp binding to M158C and I146C 1 µM of hPRLbp was flowed over a CM5 sensor chip at 50 µl/min to which 100 RU of either M158C or I146C were bound. Association was followed for 4 minutes after which buffer was flowed over the chip surface and dissociation was followed for another 4 minutes.

A RU M158C 120 100 80 R

e 60 s pon 40 s

e 20 0 -20 -100 -30 40 110 180 250 320 390 460 530 600 Time sec B G129C RU 120

100

80 R

e 60 s pons 40 e 20

-20 -100 -30 40 110 180 250 320 390 460 530 600 Time sec

(Continued)

Figure 3.10 Fits of the receptor binding curves to the cysteine mutants 50 nM, 80 nM, 100 nM, 200 nM, 500 nM and 800 nM concentrations of hPRLbp were passed over the mutants in random order at 50 µl/min. M158C (A) was fit to a two site sequential binding model, while G129C was fit to a 1:1 Langmuir binding model to determine the rate constants. The initial 3 -1 -1 -5 -1 parameters for ka1 and ka2 were set to 10 M s , kd1 was set to 10 s and kd2 was set to 10-7 s-1.

(Figure 3.10 continued)

C RU 120 K181C 100

80 Resp 60 o n

se 40

-20 -100 -30 40 110 180 250 320 390 460 530 600 Time sec

Mutant Site 1 Site 2

k k K k k K a1 d1 D1 a2 d2 D2 (M-1s-1) (s-1) (nM) (M-1s-1) (s-1) (nM) x104 x10-3 x103 x10-4

M158C 1.43±0.22 1.57±0.37 109.42 2.56± 0.46 3.91±3.86 153.11

G129C 1.96±0.09 1.19±0.11 60.54 ------

Table 3.1 Rate and equilibrium constants for receptor binding to the cysteine mutants The rate constants are an average of 3-5 experiments and the standard deviation is calculated from this data.

Experiments were carried out as described in the materials and methods to ensure that the kinetics were not influenced by mass transport limitations. 50 nM hPRLbp was flowed over the CM5 sensor chip to which 100

RU of M158C, G129C and K181C were bound at 5 µl/min, 15 µl/min and 75

µl/min. The slopes of the lines during the initial association phase at various flow rates were calculated. The average variance was about 8% and is within the acceptable range. This indicates that the association and dissociation of hPRLbp was not influenced by mass transport.

60 3.3 Discussion

The recombinant cysteine mutants can successfully be purified and refolded such that the additional cysteine is free and available for coupling to a

CM5 sensor chip. Introducing the cysteine in the binding site decreases biological activity but that it is to be expected since these are key residues for receptor binding. Two different controls (M158C and I146C) show similar binding curves demonstrating that introduction of cysteine away from the active sites do not affect the hormone activity or kinetic rate constants.

The SPR data shows that when Site 2 is corrupted the hormone still binds to the receptor via Site 1. The amount of receptor bound is less than that bound to the hormone with both binding sites available. There is about

60% of the binding compared to the wild type, which is slightly higher than the expected 50%. One possible explanation for this observation could be that once the receptor binds to the first hormone, the receptor is in a suitable conformation to bind to a second receptor via interactions at the foot region.

However, since Site 2 is not available for receptor binding, the second receptor cannot form a stable trimeric complex with the hormone and first receptor.

When Site 1 is blocked, the amount of receptor bound drops to about

5% compared to the wild type. This suggests that receptor binding at Site 2 is dependent on receptor binding at Site 1. If the differential affinity theory was correct and the two sites were independent of each other, then at saturating

61 receptor concentrations, hormone receptor complexes should still be formed through binding at Site 2, which is not observed. The little binding that is observed could be attributed to the fluid nature of most proteins. Even though

Site 1 is blocked, it is possible that as the hormone might hit the right conformation that allows for receptor binding at Site 2. However this would occur much less frequently without Site 1 bound. Receptor affinities are very similar at the two sites further suggesting that the differential affinity theory may not sufficiently explain why the two receptors bind sequentially. Also, receptor affinities at Site 1 are not affected by blocking Site 2. This suggests that Site 1 binding is independent of Site 2 availability.

A seminal paper studied binding kinetics between the rat, rabbit and bovine prolactin receptors and prolactin from various species (73). In that paper the researchers showed that hormone and receptor from the same species showed similar binding affinities for Site 1 compared to Site 2.

However, receptor from a different species than the hormone showed a higher binding affinity for Site 1 than for Site 2. Their data also showed that for hGH binding to the growth hormone receptor, blocking Site 2 using a monovalent antibody did not affect affinity at Site 1. This is consistent with the data presented here. Based on these observations the researchers proposed that

Site 1 has a higher affinity than Site 2, supporting the differential affinity hypothesis. However, the data presented here strongly suggest that the difference in affinity between the two sites when hormone and receptor from

62 different species are used is due to the inability of the hormone to restructure into the correct conformation for optimal Site 2 binding. This could be because the receptor binding epitopes for a receptor from another species may not be identical to the epitopes for a homologous receptor, preventing the right contacts between residues to induce a conformation change.

The data indicate that receptor binding at Site 2 depends on receptor binding at Site 1. One possible reason (supported by the data presented here) for sequential binding is that when the first receptor binds to Site 1, it induces a conformation change in the hormone, spatially reorganizing the residues and creating the second receptor-binding site.

CHAPTER 4

EFFECT OF CORRUPTION OF SITE 1 AND SITE 2 ON RECEPTOR BINDING

4.1 Introduction

In the previous chapter, we have demonstrated that receptor binding at

Site 2 depended on receptor occupancy at Site 1. The approach used was to completely block either Site 1 or Site 2. This was rather drastic, and the effects observed could possibly be due to a change in conformation upon being coupled to the sensor chip. We decided to mutate critical residues in either Site 1 or Site 2 to alanine to determine whether similar results would be observed. We coupled these mutations to the CM5 sensor chip with the

M158C linkage. This allowed us to determine the effects of mutations in the binding sites on receptor binding kinetics without directly blocking the binding sites and with a more subtle corruption. Since K181 and G129 worked well in the previous experiments the same residues were selected. In addition R176 was selected since this residue has also previously been identified as being important for receptor binding and biological activity (71). R176A and K181A were coupled together to make a Site 1 double mutant

4.2 Results

4.2.1 Characterization of the mutants

At least two batches of each of the mutant proteins were prepared and the yield varied between 20-30 mg per liter. The purified recombinant proteins were run on both reducing and non-reducing SDS-PAGE gels (Figure 4.1). Reducing gels showed a single band around 23 kDa in molecular weight for wild-type hPRL and the hPRL mutants. All the recombinant protein preparations thus appear to be over 95% pure. Non- reducing gels provide information about disulfide bond formation in the proteins. All the recombinant proteins appear to run at the same relative position on the non-reducing gel between 20-24 kDa indicating that disulfide bond formation was not affected by the presence of an additional cysteine.

Some dimer was observed in all the protein preparations. However the proportion of dimer is small compared to the monomeric population.

Various spectroscopic techniques were used to characterize the recombinant proteins. UV spectra of the recombinant proteins compared to the wild-type hPRL were very similar. They all had an absorption maximum at

277 nm and a 280/250 ratio between 1.75-1.9. There was no significant light scatter at 350 nm confirming the absence of aggregates or other large impurities. This indicates that disulfide bond formation was not affected by introduction of the seventh cysteine and the samples are pure (Figure 4.2 A).

65 Fluorescence spectra of the various mutants showed a maximum emission peak between 340-341 nm and were similar to the wild-type hPRL peak at 340 nm. This indicates that the hydrophobic packing of the mutants was similar to the wild type (Figure 4.2 B). Variation in the peak height is a reflection of variations in protein concentrations. CD spectra of the mutants were characteristic of α-helical proteins with two negative peaks at 222 nm and 208 nm and a positive peak approaching 190 nm. There was a slight variation in the negative peak at 208 nm (Figure 4.2 C, inset) but the difference is not significant. The helical content of the mutants does not appear to be altered relative to the wild type (Figure 4.2C). Overall, it appears that the tertiary structure of the mutant hormone preparations was not altered compared to the wild-type hPRL.

The mutants were then tested in lactogenic bioassays to compare changes in their biological activity relative to the wild-type (Figure 4.3). Again, there were no significant differences between wild-type hPRL and the M158C mutant. All three binding site mutants showed ED50 values an order of magnitude weaker than the wild-type (Table 4.1). All three binding curves were shifted to the right. K181A/M158C hPRL could reach similar maximal activity as the wild type but at much higher concentrations.

R176A/K181A/M158C hPRL and G129R/M158C hPRL had reduced maximal activity compared to the wild-type. This is not unusual since the mutations lie in the receptor binding pocket.

66 / C C A 58 8 1 1 15 8 M M 1 / / /K C R A A C M M 8 M 29 81 M 6 58 W T W 5 W 1 1 7 1 1 K W 1 M W M M M G M R M

66 66 66 45 66 45 45 36 36 36 29 45 29 24 36 29 24 20.1 24 29 20.1 24 14.2 20.1 20.1 14.2

14.2 14.2

Non Reducing Gel

/ A C 1 8C 8 8 5 5 1 1 1 K C M M / 8 C / / A 5 8 R A 6 1 M M 5 M 9 1 M 7 W T W 1 8 W 1 M M W 12 1 R M W M M G K M

66 66 66 66 45 45 45 45 36 36 36 36 29 29 29 24 29 24 24 24 20.1 20.1 20.1 20.1 14.2

14.2 14.2

14.2

Reducing Gel

Figure 4.1 SDS-PAGE gels of the hPRL mutants Samples were prepared in the absence (top) or presence (bottom) of 2- mercaptoethanol and resolved on a 15% SDS-PAGE gel. 67

0.5

0.4 ) U 0.3

0.2 Absorbance (A Wild Type hPRL 0.1 M158C G129R/M158C K181A/M158C R176A/K181A/M158C 0.0 220 240 260 280 300 320 340 Wavelength (nm)

(Continued)

Figure 4.2 Spectroscopic analysis of the hPRL mutants (A) UV Spectroscopy, (B) Fluorescence spectroscopy, (C) CD spectroscopy Samples were prepared at 25 µM (A and C) and 1 µM (B) concentrations in 10 mM Tris, pH 8.2, 150 mM NaCl. Spectra were collected at 20-25°C. The figures inset are data that are normalized to each maximal peak, 277 nm for UV, 339 nm for fluorescence and 222 nm for CD.

68 (Figure 4.2 continued)

B ) s 600 unit

y rar rbit

(a 400 y nsit e Int

200

cence Wild Type hPRL M158C G129R/M158C K181A/M158C Fluores R176A/K181A/M158C

0 300 320 340 360 380 Wavelength (nm)

C 0 Wild type hPRL M158C G129R/M158C -1

ol -5000 K181A/M158C R176A/K181A/M158C .m 2 eg.cm

d -10000 ) θ city ( i

llipt -15000 E r Mola -20000

200 210 220 230 240 250 260 Wavelength (nm)

69 120

100

80 n o i

ct 60 u

40 % Red

0.01 0.1 1 10 100 1000 10000 Hormone Dose (nM)

Wild type hPRL M158C G129R/M158C K181A/M158C R176A/K181A/M158C

Figure 4.3 Lactogenic bioassay of the hPRL mutants Increasing doses of the various binding site mutants were added to FDC-P1 cells stably transfected with the prolactin receptor. Hormone induced cell proliferation was detected using a vital dye, Alamar blue.

70 ______

Mutant ED50 (nM) Wild-type hPRL 0.83 M158C 0.80 G129R/M158C 8.96 K181A/M158C 10.71 R176A/K181A/M158C 9.70 ______

Table 4.1 ED50 values of the various hPRL mutants

4.2.2 Receptor binding to hPRL mutants with increasing disruption in Site 1

About 100 RU of M158C, K181A/M158C and R176A/K181A/M158C were coupled to the CM5 sensor chip through M158C. Various hPRLbp concentrations (50 nM, 80 nM, 100 nM, 200 nM, 500 nM, and 800 nM) were flowed over the chip surface at 50 µl/min in random order. The binding curves for all the mutants were fit to the two-site sequential binding model and is shown in Figure 4.4. In all three cases, the experimental data compared very well to the predicted model suggesting that this model accurately reflects the binding event. The residuals were less than 4 RU and chi2 values were less than 2, further supporting the use of this two-site sequential binding model.

The kinetics of hPRLbp binding to the mutant hormones were determined and the data are presented in Table 4.2. As one, and then two mutations, were placed in Site 1 the amount of receptor binding to the mutants

71 decreased. In the case of K181A/M158C, the amount of receptor bound (at a given concentration) was approximately 40% of M158C hPRL. At Site 1 both the association and dissociation rate constants were slower although the decrease was not statistically significant (p>0.05). At Site 2, the association rate constant is 3-fold slower and the dissociation rate constant is 130 fold slower (p< 0.01) as a result of the K181A mutation in Site 1 and the affinity at

Site 2 increases 50 fold. The Site 1 double mutant (R176A/K181A) showed only 20% receptor bound as compared to M158C hPRL. In this case the association rate constant at Site 1 is significantly slower, lowering the affinity at Site 1 more than 20 fold. Additionally at Site 2 the affinity increases 50 fold as a result of a significant decrease in the dissociation rate constant (kd2) by over 250 fold. The association rate constant at Site 2 (ka2) is only slightly weaker (about 5 fold). The increased affinity at Site 2 observed with both the single and double mutant does not seem to compensate for lack of receptor binding at Site 1 since the amount of receptor bound at a given concentration appears to decrease with increased corruption at Site 1. This further supports the idea that receptor binding at Site 1 is the critical step in the formation of the trimeric hPRL-hPRLbp complex.

72 RU 120 M158C 100

80 R e sponse 60

-20 -100 -30 40 110 180 250 320 390 460 530 600 Time sec

RU 120 K181A/M158C 100 80

Resp 60

o 40 n se 20 0

-20 -100 -30 40 110 180 250 320 390 460 530 600 Time sec

RU 120 100 R176A/K181A/M158C Respo 80

n 60 s e 40 20 0 -20 -100 -30 40 110 180 250 320 390 460 530 600 Time sec Figure 4.4 hPRLbp binding to the Site 1 mutants Association of various concentrations of hPRLbp from 50-800 nM was followed for 5 minutes and then dissociation followed for an additional 5 min. The binding curves were then fit to the two-site sequential binding model. The 3 -1 -1 -5 -1 initial parameters for ka1 and ka2 were set to 10 M s , kd1 was set to 10 s -7 -1 and kd2 was set to 10 s . 73 Mutant Site 1 Site 2

k k K k k K a1 d1 D1 a2 d2 D2 (M-1s-1) (s-1) (nM) (M-1s-1) (s-1) (nM) x104 x10-3 x103 x10-4

M158C 1.43±0.22 1.57±0.37 109.42 2.56±0.46 3.91±3.86 153.11

G129R/M158C 1.85±0.51 1.82±1.04 98.56 2.43±0.22 41.10±2.00 1691.36

K181A/M158C 0.34±0.08 0.56±0.22 164.51 0.88±0.80 0.03±0.03 2.91

R176A/K181A/ 0.14±0.06 3.59±0.22 2529.96 0.57±0.50 0.02±0.03 3.72 M158C

Table 4.2 Rate and equilibrium constants for hPRL binding site mutants The rate constants are an average of 3-5 experiments and the standard deviation is calculated from this data.

4.2.3 Receptor binding to a hPRL mutant with a corrupt Site 2

G129R hPRL is an established prolactin mutant that is considered a good prolactin antagonist (107,108). The binding kinetics of G129R where

Site 2 is corrupted were compared to those of M158C, where both binding sites are functional using SPR technology. The data were fit to the two site sequential binding model (Figure 4.5). The goodness of fit was based on the residuals which were less than 5 and the chi2 values, which were less than 2.

Both indicate that the experimental data fit the model well. The rate constants are shown in Table 4.2. There was no significant change in the association or dissociation rate constants or the affinity at Site 1 compared to M158C hPRL.

At Site 2, while the association rate constant was unaffected, the dissociation rate constant was 7-fold faster resulting in a 7-fold weaker affinity at Site 2.

The level of receptor binding at a given concentration was about half that of

M158C hPRL.

75 RU M158C 160

Re 110 spo

n 60 se

-40 -100 -30 40 110 180 250 320 390 460 530 600 Time sec

RU 120 G129R/M158C 100

80 Resp 60 o

n 40 se 20

-20 -100 -30 40 110 180 250 320 390 460 530 600 Time s

Figure 4.5 Binding of hPRLbp to G129R/M158C compared to M158C Association of various concentrations of hPRLbp from 50-800 nM was followed for 5 minutes and then dissociation followed for an additional 5 min. The binding curves were then fit to the two-site sequential binding model. The 3 -1 -1 -5 -1 initial parameters for ka1 and ka2 were set to 10 M s , kd1 was set to 10 s -7 -1 and kd2 was set to 10 s .

76 4.3 Discussion

Corruption of Site 1 causes a significant decrease in receptor binding and is proportional to the extent of corruption of Site 1. With K181A/M158C, the affinity at Site 1 is unaffected, and the affinity at Site 2 is increased so one would expect that there would be the same receptor binding capacity as

M158C. However the amount of receptor bound is reduced by 60%. One possible explanation is that although the change in affinity at Site 1 appears to be insignificant, the 4-fold decrease in association rate constant at Site 1 is enough for fewer receptors to bind to the hormone at Site 1. As a result, fewer receptors bind at Site 2 reducing the total binding capacity of the mutant. The increased affinity at Site 2 (the result of a decrease in the dissociation rate constant) cannot compensate for the decrease in receptor binding at Site 1.

In the case of the double mutant (R176A/K181A/M158C), the binding capacity of the mutant is reduced even further. The affinity at Site 1 is decreased as a result of an increase in the dissociation rate constant from Site

1 (Table 4.2). Once again, the increase in affinity at Site 2 cannot compensate for the detrimental effects on Site 1. This further supports the idea that receptor binding at Site 1 is necessary for receptor binding at Site 2. Attempts were made to couple the receptor to the CM5 chip using the intrinsic free cysteine to see if we could observe similar changes in receptor binding kinetics. Unfortunately, coupling the receptor completely blocked its hormone

77 binding capacity. It is possible that coupling the receptor via the cysteine limits the flexibility of the receptor rendering it incapable of ligand binding or provides a steric inhibition for hPRL binding.

In the case of the G129R/M158C mutant, the dissociation rate constant at Site 2 is 7 times faster. This decreases receptor affinity at Site 2. However, there are no changes in affinity at Site 1. In this case the receptor can bind to

Site 1 but the affinity at Site 2 is too low to hold the second receptor resulting in a 50% decrease in receptor binding compared to M158C hPRL. This supports our observation with G129C that Site 1 binding is independent of Site

2 binding. In conclusion, the results from the binding site mutants are in complete agreement with the results from the binding site cysteine mutants discussed in Chapter 3 and further support our observation of communication between the two receptor binding sites.

CHAPTER 5

CONFORMATION CHANGE INDUCED BY RECEPTOR BINDING

5.1 Introduction

In the previous two chapters functional coupling of the receptor binding sites on hPRL were examined using surface plasmon resonance technology.

The studies have shown that receptor binding at Site 1 on hPRL is independent of a functional site 2. However Site 2 receptor binding depends on a functional Site 1. Even a small corruption in Site 1 significantly affects its receptor binding capacity. The next question that should be addressed is the mechanism by which the binding sites communicate with each other. In the case of hGH, crystal structures have clearly demonstrated a conformation change in the hormone upon receptor binding. Some of the site 1 binding- induced changes observed include an extension of helix 2 into the loop between helix 2 and helix 3, and the formation of mini-helix 1 in the unstructured loop between helix 1 and 2. In the absence of crystal structures of hPRL in the free and receptor bound forms, other approaches can be used to determine if there is a conformation change. Fluorescence resonance energy transfer (FRET) was used to test whether receptor binding induces a

79 conformation change in the hormone. In this case, the hormone can be labeled with an extrinsic fluorochrome and the efficiency of fluorescent energy transfer from an intrinsic fluorochrome such as tryptophan can be determined.

The extrinsic fluorochrome used in this case is CPM whose structure is drawn below. The absorption maximum is at 385nm and the emission maximum is at

469 nm. CPM forms a thioether bond with the sulfhydryl group of the cysteine.

7-diethylamino-3-(4'-maleimidylphenyl)- 4-methylcoumarin (CPM)

Maleimides react with sulfhydryl groups by the following reaction:

(From www.molecularprobes.com)

Energy transfer from tryptophan residues to CPM coupled to the free cysteine in M158C can be used to monitor conformation changes occurring in hPRL upon receptor binding.

80 5.2 Results

5.2.1 Absorption spectra of proteins labeled with CPM

Wild-type hPRL (with three disulfide bonds and no free cysteine) and

M158C (with one free cysteine) hPRL were labeled with CPM. Labeling of the proteins was verified by mass spectrometry. In both cases, about 25% of the protein was labeled. A peak with a molecular weight corresponding to a single label was the predominant labeled form. A small peak corresponding to two labels was also observed. Since wild-type hPRL has no free cysteine, it suggests that one of the three disulfide bonds was broken or labeling occurred at other sites. Previous studies on ovine PRL have demonstrated that breaking the N-terminal and C-terminal disulfide bond do not significantly affect the biological activity of the hormone (55). DTT concentrations in the 30 mM range were required to break the central disulfide bond that was critical for activity. In this case, it is unlikely that the large disulfide loop was affected.

The UV absorption spectra of labeled and unlabeled wild-type hPRL and

M158C were compared to verify that the tertiary structure of the protein was not affected by the labeling process (Figure 5.1). Only the labeled proteins have an absorption peak around 389 nm corresponding to the absorption of

CPM. All the proteins have a peak around 280 nm corresponding to the absorption peak for the aromatic amino acids. The peak is shifted near 270 nm in the labeled proteins. The unlabeled proteins have a characteristic

280/250 ratio around 2. However this ratio is lowered to about 1 in the labeled

81 proteins due to an increased absorption at 250 nm. This suggests that the labeling has placed a strain on one or more of the disulfide bonds. Based on the mass spectrometry data, it does appear that one disulfide bond might be broken. This might place a strain on the other disulfide bonds. The absorption spectrum also shows that labeling of both proteins was equivalent since the magnitude of the CPM absorption peak was similar for both proteins. The absorption spectrum of hPRLbp was also recorded to ensure that the receptor was folded correctly. The receptor also showed an absorption maximum around 280 nm and a 280/250 ratio of 2. The magnitude of the hPRLbp peak is higher than that of the hormone at the same concentration because hPRLbp contains ten tryptophan residues as opposed to two in the hormone.

5.2.2 Fluorescence Resonance Energy Transfer (FRET)

1 µM of the labeled wild-type hPRL and M158C hPRL were incubated with increasing concentrations of hPRLbp and allowed to reach equilibrium.

The intrinsic tryptophan residues were selectively excited at 295 nm, and emission spectra collected from 300 nm to 570 nm (Figure 5.2). CPM emission maximum is around 469 nm, while tryptophan emission maximum is around 340 nm. In both proteins, the magnitude of the peak at around 469 nm

(corresponding to the emission maximum of CPM) increases as the receptor concentration increases. This suggests that there is an increase in energy transfer from tryptophan residues to CPM. The transfer of energy could come

82 from the tryptophan residues in the hormone or the receptor. The model of rabbit prolactin bound to two rabbit prolactin receptors (109) shows that the tryptophan residues in the receptor are at least 10 Ǻ or more further away from

CPM than the two intrinsic tryptophans in the hormone, if the hormone is labeled at 158. Therefore, very little energy contribution comes from the receptor tryptophans. This increased fluorescence of CPM can be attributed to an increased transfer of energy from the intrinsic tryptophans. The simplest explanation for this energy transfer is that there is a conformation change in the hormone upon receptor binding that is bringing the two fluorochromes closer together. Although both wild-type hPRL and M158C hPRL are equally labeled, it appears that the energy transfer in the case of M158C hPRL is much stronger than with wild-type hPRL (Table 5.1). It is possible that in the case of the wild-type hPRL, the label is on one of the cysteine groups of the broken disulfide bond. This could weaken receptor binding and thus result in a lower efficiency of energy transfer. In the case of M158C hPRL, the free cysteine could be predominantly labeled. As a result the receptor binding sites are not affected and the efficiency of energy transfer is higher. 50-fold excess of unlabeled hormone was added to the along with labeled M158C hPRL and receptor and this resulted in a decrease in CPM fluorescence (Figure 5.3). The unlabeled hPRL successfully competed for some of the receptor binding sites in the labeled M158C demonstrating that the receptor binding is specific.

83 0.6 hPRLbp WT hPRL 0.5 WT hPRL + CPM M158C M158C + CPM

) 0.4 U

0.3 bance (A sor b

A 0.2

0.1

0.0 250 300 350 400 450 500 Wavelength (nm)

Figure 5.1 Absorption spectra comparing labeled and unlabeled proteins hPRLbp, labeled or unlabeled wild-type hPRL (WT hPRL) and M158C were diluted to 1 µM concentration in PBS and absorption spectra collected from 220-500 nm.

84 600 hPRLbp 1µM WT 500 1µM WT+1µM hPRLbp

units) 1µM WT+2µM hPRLbp y 1µM WT+5µM hPRLbp

itrar 400 b r a y ( t i 300

e Intens 200 cenc es 100 uor l F

0 300 350 400 450 500 550 Wavelength (nm)

600 1µM hPRLbp 1µM M158C

its) 1µM M158C+1µMhPRLbp

un 1µM M158C+2µM hPRLbp 500 y

r 1µM M158C+5µM hPRLbp a rbitr

a 400 ( ity

ns 300

200 ence Inte c

100 Fluores

0 300 350 400 450 500 550 Wavelength (nm)

Figure 5.2 Fluorescence spectra of labeled proteins mixed with increasing receptor concentrations 1 µM hormone was mixed with increasing receptor concentrations and allowed to come to equilibrium for 1 hour at room temperature. Samples were then excited at 295 nm and fluorescence spectra collected from 300-570 nm.

85 1000 s) it 800 ry un a r it rb

a 600 y ( it s n e t

n 400 e I c n sce

e 200 r o Flu

0 350 400 450 500 550 Wavelength (nm)

1µM M158C 1µM M158C + 1µM hPRLbp 1µM M158C + 2µM hPRLbp 1µM M158C + 1µM hPRLbp + 50µM WT hPRL

Figure 5.3 Competition experiment using excess unlabeled hPRL 1 µM M158C hPRL was incubated with increasing receptor concentrations or a 50-fold molar excess of unlabeled wild-type hPRL. Emission spectra were collected from 300-570 nm. Fluorescence intensities in this experiment are higher because almost 50 % of the sample used was labeled.

Sample CPM Maximum Area Under the Curve Difference Intensity (420 nm-550 nm)

WT hPRL 197.8 27276 -

WT hPRL+1µM hPRLbp 214.3 28909 1633

WT hPRL+2µM hPRLbp 231.6 31699 4423

WT hPRL+5µM hPRLbp 256.8 36092 8816

M158C 181.1 23954 -

M158C+1µM hPRLbp 220.5 29687 5773

M158C+2µM hPRLbp 257.8 35163 11209

M158C+5µM hPRLbp 302.8 42202 18248

Table 5.1 Increases in fluorescence intensity of CPM in labeled hormones upon receptor binding

87 5.3 Discussion

FRET was used to determine whether there was a conformation change in the hormone upon receptor binding. There appears to be some strain in the disulfide bonds of the labeled hormones. However this does not appear to affect receptor binding since there was an increase in fluorescence of CPM as increasing concentrations of receptor were added to the labeled hormone. This indicates that there was clearly some interaction between the hormone and the receptor resulting in the two tryptophan residues in the hormone being reoriented closer to the extrinsic fluorochrome. The ability of a

50-fold excess of unlabeled wild-type hPRL clearly demonstrates that there is specific binding between the labeled hormone and the receptor. The question that remains to be addressed is the relative contributions of the tryptophan residues from the hormone and the receptor. The shortest distance between one of the terminal cysteines and a tryptophan residue in the receptor (based on the rabbit PRL model) is 24 Ǻ, while the farthest distance between the terminal cysteine and a tryptophan in the receptor is 12 Ǻ. Whether the label was at the free cysteine in M158C hPRL or on one of the cysteines of the broken disulfide bond, the label is much closer to the two intrinsic tryptophan residues in the hormone than the tryptophans in the receptor. Thus the major energy contribution must come from the tryptophans in the hormone. Tryptic digests of the labeled wild-type hPRL and M158C hPRL followed by mass spectrometry could resolve the issue of the location of the label in these two

88 proteins. Crystal structures of free and receptor-bound forms of hPRL would be the best answer to the question of conformation change in hPRL upon receptor binding.

As an alternative approach CD spectroscopy of the hPRL – hPRLbp complex was compared to the sum of the spectra of the free hormone and receptor. No differences in molar ellipticity were observed. This suggests that there were no detectable changes in the helical content of the hormone. It is possible that the instrument is not sensitive enough to detect very small changes in helicity or that the helical content does not change as part of a binding-induced conformation change.

CHAPTER 6

IDENTIFICATION OF RESIDUES CRITICAL FOR PROLACTIN ACTIVITY

6.1 Introduction

In the previous chapters, hPRL-hPRLbp interactions have been studied.

The studies have shown that receptor binding is sequential, both receptor binding sites have similar affinities, and increasing receptor affinity at Site 2 cannot compensate for corruption of site 1 supporting the idea that receptor binding at Site 1 is the crucial step in hPRL receptor interactions. Studies also strongly suggest that there is a conformation change in hPRL upon receptor binding. The next step was to identify residues in hPRL that might transmit the conformation change from Site 1 to Site 2. Previous studies on hGH have demonstrated that there is a hydrophobic motif distal to the two receptor binding sites that might be responsible for transmitting this conformation change (110). Five residues lying in mini-helix 1 (F44), helix 2 (L93), and helix

4 (Y160, L163, Y164) were all demonstrated to affect receptor binding at Site

2 when mutated to glutamate. When the amino acid sequence of hPRL was compared to that of hGH, it was observed that there were regions of hydrophobic residues located in areas analogous to the critical hydrophobic

90 residues in hGH (Figure 6.1). These hydrophobic residues were mutated to either aspartate or glutamate to disrupt hydrophobic packing and the effect on hPRL biological activity was tested. There are several reasons for substitution of charged residues instead of alanine or glycine. All these residues are located in helices. Substitution with a glycine could disrupt helix formation and result in a global effect rather than a local effect. Although alanine is used frequently in site-directed mutagenesis, the methyl side chain makes it hydrophobic. Substitution with alanine will reduce the bulk of the side chains, but may not significantly alter the hydrophobic interactions between residues.

To avoid these problems, negatively charges residues were substituted for the hydrophobic residues.

Studies have shown that deletion of a portion of mini-helix 1 (the loop between helix 1 and helix 2) distal to the receptor binding sites causes a

10,000-fold decrease in biological activity (Peterson and Brooks, manuscript in preparation). This mutant (called ∆41-52 hPRL) induces apoptosis in breast cancer cell lines (Almgren and Brooks, manuscript in preparation). Each residue in the deleted segment of mini-helix 1 was individually mutated to determine which residues were critical to hPRL activity. In mini-helix 1, hydrophobic residues were substituted with negatively charged residues

(T45E, F50E, I51E, and A54D), glycine was substituted with phenylalanine

(G47F, G49F) to introduce bulk and positively charged residues were substituted with alanine (H46A, R48A) or negatively charged residues (K53E).

91 The lactogenic bioassay used to test the biological activity of recombinant proteins can provide information about the steric and mechanistic factors that contribute to receptor binding at Site 1 and Site 2. The assay was first developed to test the ability of various hGH antagonists to bind to the growth hormone receptor. In this assay, cells stably transfected with the prolactin receptor are treated with increasing concentrations of recombinant hormone preparations. The lactogenic hormones show a characteristic bell- shaped dose-response curve. As the hormone concentration increases, active hormone-receptor complexes are formed, inducing cell proliferation which is reflected in the agonist phase of the dose-response curve. The ED50 value is a reflection of both Site 1 and Site 2 binding affinity. As the hormone concentration continues to increase, all the receptors are occupied and saturation is reached. Changes in the maximum response are a reflection of

Site 2 activity. As more hormone is added, the equilibrium shifts in favor of forming 1:1 hormone receptor complexes and self-antagonism is observed.

Mutations in Site 1 shift both the agonist and antagonist phase of the curve.

Mutations in Site 2 change the maximal response. The ID50 values are a reflection of receptor binding via Site 1 and therefore a measure of Site 1 affinity. A shift in the agonist phase of the dose-response curve without a concurrent shift in the antagonist phase indicates that the mutation affects binding at Site 2. Based on this interpretation of this assay, the effect of mutations in hPRL not directly in the receptor-binding interfaces can be

Helix 1 LPICPGGAARCQVTLRDLFDRAVVLSHYIHNLSSEMFSEFDKRYT- VQTVP------LSRLFDHAMLQAHRAHQLAIDTYQEFEETYIP

HGRG—-FITKA-INSCHTSSLATPEDKEQAQQMNQKDFLSLIVSI KDQKYS-FLHDSQTSFCFSDSIPTPSNMEETQQKSNLELLRISLLL

Helix 2 Helix 3 LRSWNEPLYHLVTEVRGMQEAPEAILSKAVEIEEQTKRLLEGMEL IESWLEPVRFL-RSMFANNLVYDTSDSDDYHLLKDLEEGIQTLMG

Helix 4 IVSQVHPETKENEIYPVWSGLPSLQMADEESRLSAYYNLLHCLRR RLEDGSRRTGQILKQTYSKFDTNSHNHDALLKN---YGLLYCFRK

DSHKIDNYLKLLKCRIIHNNNC DMDKVETFLRMVQCRS-VEGSCGF

Figure 6.1 Comparison of the primary amino acid sequence of hPRL (top line) and hGH (bottom line) Residues in green have previously been identified as important for hGH activity. The residue in blue (hGH L163E) was identified as critical for the structure of hGH. Residues marked in red are the ones mutated in this study. Underlined sequences are the putative helices in hPRL.

93 attributed to effects on either Site 1 or Site 2. Thus, site-directed mutagenesis in conjunction with this lactogenic bioassay is an appropriate experimental approach to discern the motif required for the functional linkage of the two binding sites in hPRL.

6.2 Results

6.2.1 Characterization of the mutants in Helix 2 and Helix 4

The following mutants were prepared: L95E, Y96E, H97E, L98E, V99D,

V102D in Helix 2; L165E, Y168E, Y169E, L171E, L172E, H173E in Helix 4.

Since the four helices run in an up-up-down-down configuration, the residues all lie distal to the receptor binding sites (Figure 6.12). The recombinant proteins were expressed and purified as described in Chapter 2. L172E could not be purified despite several attempts. It is possible that this residue is a structural determinant and mutating it could affect the tertiary structure of the protein and hence its ability to be purified by anion exchange chromatography.

The proteins were all run on SDS-PAGE gels to ensure purity and correct molecular weight (Figure 6.2). The reducing gel indicates that the proteins were over 95% pure and the molecular weight was indistinguishable from the wild-type hPRL. The non-reducing gel shows that the samples were largely monomeric with no significant effects on disulfide-bond formation compared to the wild-type hPRL.

94 The proteins were characterized by various spectroscopic techniques to ensure that they were folded correctly. UV absorption spectroscopy showed that several mutants (L95E, L98E, V99D, Y168E and L171E) showed shifts in the wavelength of maximum absorption relative to wild-type hPRL (Figure 6.3).

Maximum absorption by wild-type hPRL was seen at 280 nm. The mutants listed above all had a maximum absorption around 276-277 nm. More than a

2 nm shift in the peak is considered significant. These same mutants also showed a significant decrease in their 280/250 ratio. Ratios varied from 1.4-

1.8 for the mutants compared to a 280/250 ratio of 2.2 for the wild-type hPRL.

This suggests that these mutations are affecting the environment around the tryptophan residues and are putting a strain on one or more of the disulfide bonds. This is corroborated by observations from fluorescence spectroscopy of a shift in the maximum emission peak by more than 2 nm for these mutants

(Figure 6.4). L98E and V99D showed the maximum shift of 4-5 nm. This clearly suggests that the environment surrounding the reporting tryptophans is being altered in these mutants. In addition H97E and H173E show a very slight shift in the wavelength of maximum fluorescence emission without any significant effects on the UV absorption spectrum. All the other mutants show the characteristic emission maximum around 340 nm. CD spectra of all the proteins show a characteristic negative peak at 222 nm. The CD spectrum of

Y96E and L98E show a significant variation from the spectrum of the wild-type protein at 208 nm suggesting that there is a change in the electronic

95 environment around one or more of the helices (Figure 6.5). It is interesting that Y96E does not show any variation in the UV absorption or fluorescence spectrum and only in the CD spectrum.

6.2.2 Biological activity of the helix 2 and helix 4 mutants

The activity of the mutants was tested in lactogenic bioassays as previously described to test the effect of the mutations on biological activity

(Figure 6.6). The ED50 values are shown in Table 6.1. The mutants appear to fall into three distinct groups. The dose-response curve is completely shifted to the right in the case of L95E, V99D, L165E, Y169E and H173E. In addition, the peak height increases between 1.2-1.5 times that of the wild-type hPRL.

This suggests that Site 1 affinity is decreased while Site 2 affinity is increased.

V99D and H173E appear to have the most dramatic changes in ED50 values and maximum response. L165E and Y169E show moderate changes in ED50 values but very significant increases in maximum response. The second group includes L98E and L171E. Both appear to show right shift of the agonist phase of curve and an increase in peak height, but no change in the antagonist phase. In this case, Site 1 appears to be unaffected since the antagonist phase is not shifted. The effects on Site 2 appear to be contradictory. The right shift in the agonist phase suggests that Site 2 activity is decreased but the increase in maximal response indicates that Site 2 activity increases. The third group includes Y96E, H97E, V102D and Y168E.

96 These mutants do not show any changes in the agonist or antagonist phase of the curve but do show a moderate increase in peak height suggesting that Site

1 affinity is unchanged but Site 2 affinity is moderately increased. The dose- response curves for L95E and V99D are not shown here since the experiments were performed independently. It appears that all the mutations in helix 2 and 4 alter the activity of hPRL. Their effects vary and no correlation is observed between structural changes and biological activity.

6.2.3 Characterization of the mini-helix 1 mutants

The mini-helix 1 mutant proteins were resolved on SDS-PAGE gels under reducing and non-reducing conditions and appear to be over 95% pure and at the correct molecular weight compared to the wild-type (Figure 6.7). All the mutants showed a UV absorption maximum between 279-280 nm (Figure

6.8). No light scattering was detected at 350 nm in the UV spectra and the

280/250 ratios of most of the mutants were between 2.0-2.2. The only exception was G47F which had a 280/250 ratio of 1.9. Introducing a bulky group like phenylalanine in place of a glycine at this position could put a strain on one or more of the disulfide bonds. Fluorescence spectra of the proteins showed an emission maximum between 340-342 nm. G49F and F50E showed a slight shift in the maximum wavelength to 344 nm. Since phenylalanine makes a small contribution to the fluorescence signal, adding or removing Phe at either of these two positions could affect the emission

97 maximum. The CD spectrum of H46A showed significant variation in the 208 nm region suggesting a change in the environment around one or more of the helices. The magnitude of the CD spectrum of H46A was also significantly lower compared to the wild type. Overall, it appears that all the mini-helix 1 mutants are folded similar to the wild-type with very subtle effects on the structure.

6.2.4 Biological activity of mini-helix 1 mutants

The mini-helix 1 mutants were tested in lactogenic bioassays to determine the effect of the mutations on biological activity. All the mutants show a right shift in the agonist phase of the curve. ED50 values could not be determined since maximum response was not reached. But it appears that all these residues are important for hPRL activity. However it does appear that the F50E mutation causes a right shift in the antagonist portion of the curve and an increase in Site 2 activity. All the other mutations cause a right shift in the dose response curve. It is unclear from the data available whether there is a change in the antagonist phase or the maximum response.

98 2 5 8 9 1 3 WT 95 96 97 98 99 10 16 16 16 17 17

45 36

29 24

20.1

14.2

Reducing gel

5 1 2 8 9 3

WT 95 96 97 98 99 10 16 16 16 17 17

45 36 29 24

20.1

14.2

Non-reducing gel

Figure 6.2 SDS-PAGE gels of mutants in helix 2 and helix 4 15-20 µg of the recombinant protein preparations were resolved on a 15% SDS-PAGE gel in the presence (top panel) or absence (bottom panel) of 2- mercaptoethanol 99 1.0

0.8 U) 0.6 (A ce n

a wild type hPRL

rb L95E 0.4 so Y96E b H97E A L98E V99D 0.2 V102D

0.0 220 240 260 280 300 320 340 Wavelength (nm)

1.0

0.8 U) 0.6 ce (A

an wild type hPRL 0.4 L165E sorb Y168E

Ab Y169E L171E H173E 0.2

0.0 220 240 260 280 300 320 340 Wavelength (nm)

Figure 6.3 Absorption spectra of helix 2 (top) and helix 4 (bottom) mutants Samples were prepared at 25 µM concentrations in 10 mM Tris, pH 8.2, 150 mM NaCl. Spectra were collected at 25°C. Panel inset is data normalized to 277 nm. 100 400 wild type hPRL

) L95E Y96E H97E L98E

ry units 300

a V99D

itr V102D b r a y ( t 200

100 uorescence Intensi Fl

0 300 320 340 360 380 400 Wavelength (nm)

wild type hPRL

) L165E 300 Y168E Y169E

ry units L171E a H173E itr b r a 200 y ( t

100 uorescence Intensi Fl

0 300 320 340 360 380 400 Wavelength (nm)

Figure 6.4 Fluorescence spectra of helix 2 (top) and helix 4 (bottom) mutants Samples were prepared at 1 µM concentrations in 10 mM Tris, pH 8.2, 150 mM NaCl. Spectra were collected at 25°C. Panel inset is data normalized to 340 nm.

101 0 wild type hPRL L95E Y96E H97E -1 L98E

ol V99D V102D .m 2 -10000 ) deg.cm θ

-20000 city (

Molar Ellipti -30000

200 210 220 230 240 250 260 Wavelength (nm)

0 wild type hPRL L165E Y168E

-1 -5000 Y169E ol L171E .m

2 H173E -10000 ) deg.cm

θ -15000

-20000 lar Ellipticity ( o

M -25000

-30000 200 210 220 230 240 250 260 Wavelength (nm)

Figure 6.5 CD spectra of helix 2 (top) and helix 4 (bottom) mutants Samples were prepared at 25 µM concentrations in 10 mM Tris, pH 8.2, 150 mM NaCl. Spectra were collected at 25°C. Panel inset is data normalized to 222 nm. 102 140 wild type hPRL Y96E H97E 120 L98E V102D

100

tion 80 educ

R 60 %

0.01 0.1 1 10 100 1000 10000 Hormone Dose (nM)

wild type hPRL 250 L165E Y168E Y169E 200 L171E H173E

150 tion educ

R 100 %

0.01 0.1 1 10 100 1000 10000 Hormone Dose (nM)

Figure 6.6 Lactogenic assay of helix 2 (top) and helix 4 (bottom) mutants Increasing hormone doses were incubated with FDC-P1 cells stable transfected with the prolactin receptor. Cell proliferation was assessed using a vital dye. The experiment was performed three times and representative data are presented here. 103

______

Mutant ED50 (nM)

Wild-type hPRL 0.72

L95E 5.6

Y96E 0.45

H97E 0.59

L98E 63.13

V99D N/Da

V102D 0.75

L165E 29.50

Y168E 0.66

Y169E 64.80

L171E 5.7

H173E 1130 ______a ED50 values could not be determined since saturation was never reached

Table 6.1 ED50 values of helix 2 and helix 4 mutants

104

WT 45 46 47 48 49 50 51 53 54

45 36

29 24

20.1

14.2

Non-reducing gel WT 45 46 47 48 49 50 51 53 54

45 36

29 24

20.1

14.2

Reducing gel

Figure 6.7 SDS-PAGE gels of mini-helix 1 mutants 15-20 µg of the recombinant protein preparations were resolved on a 15% SDS-PAGE gel in the absence (top panel) or presence (bottom panel) of 2- mercaptoethanol

105

0.8

0.6 )

0.4 rbance (AU o Abs

0.2

0.0 220 240 260 280 300 320 340 Wavelength (nm)

Wild type hPRL T45E H46A G47F R48A G49F F50E I51E K53E A54D

Figure 6.8 Absorption spectra of mini-helix 1 mutants Samples were prepared at 25 µM concentrations in 10 mM Tris, pH 8.2, 150 mM NaCl. Spectra were collected at 25°C. Panel inset is data normalized to 277 nm.

106 400 units)

300 itrary b r a

200 tensity ( ce In n e

c 100 s e r u Flo

0 300 320 340 360 380 400 Wavelength (nm)

wild type hPRL T45E H46A G47F R48A G49F F50E I51E K53E A54D

Figure 6.9 Fluorescence spectra of mini-helix 1 mutants Samples were prepared at 1 µM concentrations in 10 mM Tris, pH 8.2, 150 mM NaCl. Spectra were collected at 25°C. Panel inset is data normalized to 340 nm.

107

-1 -5000 ol .m 2

cm -10000 ) deg. θ -15000 ( y cit i

llipt -20000 E

Molar -25000

-30000 200 210 220 230 240 250 260 Wavelength (nm)

wild type hPRL T45E H46A G47F R48A G49F F50E I51E K53E A54D

Figure 6.10 CD spectra of mini-helix 1 mutants Samples were prepared at 25 µM concentrations in 10 mM Tris, pH 8.2, 150 mM NaCl. Spectra were collected at 25°C. Panel inset is data normalized to 222 nm.

108

200 wild type hPRL T45E H46A G47F 150 R48A G49F F50E I51E K53E 100 A54D

d 50 t i

0 0.1 1 10 100 1000 10000

Hormone Dose (nM)

Figure 6.11 Lactogenic bioassay of mini-helix 1 mutants Increasing hormone doses were incubated with FDC-P1 cells stable transfected with the prolactin receptor. Cell proliferation was assessed using a vital dye. The experiment was performed three times and representative data are presented here.

109 6.3 Discussion

All the hPRL mutants appeared to be over 95% pure and have the right molecular weight based on the SDS-PAGE gels. Spectroscopic studies suggest that some of the mutations have affected the tertiary structure of the hormone and this could influence their biological activity. L98E, V99D, Y168E, and L171E all showed changes in their UV and fluorescence spectra relative to the wild type hormone. Y96E, L98E and H46A all show changes in the CD spectra at 208 nm. These three mutants also show the largest changes in magnitude of the CD spectra. Not all the mutants show changes in their structure based on spectroscopic analysis. However, they all show changes in biological activity. Some are more dramatic than others but all are significant and reproducible. As mentioned in the results, all the helix 2 and helix 4 mutants fall into three groups. The mutants that show structural changes based on spectroscopic analysis are distributed between these three categories. There does not appear to be a correlation between the structural changes and the pattern of change in biological activity.

L98E and L171E show a right shift in the agonist phase and an increase in the maximum response. The lack of change in antagonist activity suggests that Site 1 activity is unaffected. The increase in maximum response suggests that Site 2 activity is increased. It is unclear then what causes the right shift in the agonist phase of the dose-response curve. Based on our theoretical model of hPRL, these two residues articulate closely within the

110 four-helix bundle (Figure 6.13 A). Mutating either one of these to glutamate could disrupt the hydrophobic packing and cause a conformation change in the molecule affecting the activity at Site 1 and 2.

Y96E, H97E, V102D and Y168E all cause an increase in peak height without affecting the agonist or antagonist phase, suggesting that only Site 2 activity is affected. In the theoretical model, the side chains of these residues extend out to the groove between helix 2 and 3. Y168 articulates closely with

F40 and F44 in mini-helix 1 (Figure 6.13 C). Mutating these residues could change the position of helix 3 relative to helix 1, affecting site 2 activity. Y96 lies in the groove between helix 2 and 3. Mutating it to a glutamate could repel helix 3 away from helix 2, reorienting it closer to helix 1 increasing affinity at

Site 2.

L165E, Y169E and H173E all shift the entire bioassay curve coupled with an increase in the peak height. This suggests that the effects of these mutations are on both Site 1 and Site 2 – decreasing Site 1 activity but increasing Site 2 activity. The side chains of these residues appear to extend out between helix 1 and 4. Y169E and H173 appear to form part of a stack of aromatic residues along with F37 in helix 1 and F50 in mini-helix 1 Figure 6.13

B). Substitution of a glutamate at Y169, H173 or F50 could disrupt these interactions repelling helix 1 away from helix 4 and causing a decrease in Site

1 activity. However, movement of helix 1 away from 4 could orient it such that it is in closer proximity to helix 3 thus increasing Site 2 affinity. This is in

111 agreement with the observed results for each of the mutations. Mutations of each of the residues in mini-helix 1 cause a right shift in the agonist phase of the curve. Mini-helix 1 fits into the groove between helix 2 and helix 4. It is possible that mutations could disrupt the interactions between helix 2 and 4 thus indirectly affecting Site 1 binding.

L95, V99 and V102 appear to articulate closely with other hydrophobic residues in helix 3 and the loop between helix 3 and 4. Again introducing charge into these positions could disrupt the hydrophobic packing between these residues and affect the receptor binding sites.

The discussion above is based on a theoretical model of hPRL and may not reflect the exact role of each of these hydrophobic residues for the activity of hPRL. But what is clear is that these hydrophobic residues are critical for optimal hPRL activity. Based on the influence of each of the hydrophobic residues on Site 1 and Site 2 activity, hPRL might use a mechanism slightly different from hGH for receptor binding. In the case of hPRL, there may not be a significant restructuring of the hormone upon receptor binding at Site 1. The hydrophobic residues could already be packed in a cluster distal to the receptor binding sites. Movement of the helices relative to one another could place the binding epitopes in helix 1 and helix 3 in the appropriate conformation to create a functional Site 2. This suggests that although the overall mechanism of receptor binding might be similar among the lactogenic hormones, the detailed mechanics of receptor binding are clearly different.

112 Kinetic analysis of prolactin receptor binding by these mutants will provide a better understanding of the impact of the mutations on receptor binding affinities at Site 1 and Site 2.

113 Site 1 Site 2

Figure 6.12 Position of all the residues mutated in mini-helix 1 (red), helix 2 (blue) and helix 4 (green) The figure was generated using the theoretical model of hPRL generated using Swiss Modeler.

114

L171

L98

(Continued)

Figure 6.13 Articulation between hydrophobic residues in the hPRL model (A) L98 and L171 pack into the core of the four-helix bundle (looking down the core of the four-helix bundle) (B) Stacking of F37, F50, Y169 and H173 between helix 1, helix 4 and mini- helix 1 connecting helix 1 and 2 (C) Packing of F40, Y44 and Y168 between helix 1 and helix 4 115

(Figure 6.13 Continued)

F50

Y169 F37

H173

Site 2 Site 1

(Continued)

116 (Figure 6.13 continued)

Y44

Y168

F40

Site 2

Site 1

117

CHAPTER 7

DISCUSSION AND PERSPECTIVES

The goal of the work presented here was to determine the mechanism of prolactin receptor binding by human prolactin. Binding studies using prolactin blocked at either Site 1 or Site 2 and saturating receptor concentrations clearly show that receptor binding is sequential. Site 1 binding is independent of a functional Site 2, but Site 2 binding requires receptor binding at Site 1. These results are further supported by kinetic studies where receptor association and dissociation rates at Site 1 are not affected by blocking Site 2. In addition, the kinetic studies using M158C show that the affinities at the receptor binding sites are very similar. This is in complete agreement with previously published work using hormone and receptor from homologous species where affinities were determined for rat, rabbit and bovine prolactin receptors binding to hPRL from a variety of species (73).

Kinetic studies using Site 1 and Site 2 mutants demonstrated that corruption of

Site 1 significantly affected the receptor binding capacity of the hormone.

Even an increase in Site 2 affinity could not compensate for a decrease in Site

1 affinity further supporting idea that Site 1 binding is critical for receptor 118 binding at Site 2. Corruption of Site 2 did not affect binding rate constants at

Site 1 further supporting the independence of Site 1 binding from Site 2.

Since crystal structures of hGH demonstrate an increase in helical content both in helix 2 and mini-helix 1 (52,59) upon receptor binding, CD spectroscopy was used to compare the molar ellipticity of free hPRL and hPRLbp versus the molar ellipticity of receptor bound to the hormone.

However, no changes could be detected. The helical changes may be too small and detection might be limited by the sensitivity of the instrument.

Absence of a change does not imply that there is no conformation change at all. FRET experiments strongly suggest that a conformation change does take place in the hormone as a result of receptor binding. However, the nature of this conformation change is unclear at the present time. In hPRL, the articulation of the helices may be important while the formation or loss of helical content may not be central to the mechanism. Clearly, structural studies are necessary to resolve this point.

Studies on hGH have also identified a hydrophobic motif distal to the receptor binding sites that are critical for hGH binding to the prolactin receptor but not for hGH binding to the somatotrophic receptor (110). This motif is critical for the functional coupling of Site 1 and Site 2. Site-directed mutagenesis was used to determine if a similar motif was required in hPRL.

Unlike hGH, all hydrophobic residues mutated in hPRL appeared to influence activity. This suggests that receptor binding mechanisms might differ among

119 the lactogenic hormones. hGH binds to both the prolactin and growth hormone receptor while hPRL only binds to the prolactin receptor. It is possible that hGH requires more flexibility to accommodate two different receptors while hPRL does not. In hPRL, the hydrophobic residues might already be packed into a motif and receptor binding at Site 1 might move the helices by changing their articulation relative to the helix bundle and reorient them to the right conformation to create Site 2. Based on these results it is clear that the two receptor binding sites are functionally coupled. In terms of receptor binding kinetics, this coupling is unidirectional. Site 1 influences Site

2 but Site 2 does not affect Site 1. However a functional Site 2 is required for a biologically active trimeric complex to form. Thus, the idea of functional coupling between allosteric sites on a molecule that was proposed 50 years ago for oxygen binding to hemoglobin can be applied to interactions between lactogenic hormones and their receptors.

Several questions remain to be addressed. Although several hydrophobic residues have been identified as important for hPRL activity the effect of these mutations on receptor binding affinities is unclear. Kinetic studies using surface plasmon resonance can help address that question.

NMR or crystal structures of hPRL free in solution and bound to the receptor will be the ideal next step to confirm the details of the mechanism of receptor binding. These structures can help address the question of conformation change and the role of the hydrophobic residues in receptor binding.

120 After the preparation of this document, the solution structure of free hPRL was published (111). Overall, the model used in this work is very similar to the NMR structure (Figure 7.1). The helical regions only differ by 1-2 amino acids. The four-helix bundle does appear to be more structured in the solution structure as compared to the model. This could support the theory that hPRL does not need as much flexibility as hGH which has to accommodate two receptors. However, there are some differences in the orientation of the functional groups. Some of the hydrophobic residues described in this work appear to interact differently with each other. For example, Y168 is oriented towards Y169, H173 and F37 rather than Y44 and F40. However, the idea that these hydrophobic residues still articulate closely with each other still holds true.

Several studies in hGH (61) and ovine placental lactogen (oPL) (53) show that receptor binding at Site 2 in hGH or oPL requires contacts between the two foot domains (D2) of the two receptors. It is believed that most of the free energy for the trimeric complex formation comes from the foot domain interactions. No such studies have so far been performed on hPRL. Our binding studies using saturating receptor concentrations suggest that receptor- receptor interactions might be taking place, but a functional Site 2 is required for a stable trimeric complex formation. The rate constants obtained for the second binding site are really a combination of the rate constants describing the coordinated interaction between the second receptor and Site 2 and the

121 interaction between the two foot domains of the receptor. It would be interesting to determine the exact contributions from each of these reactions.

It is possible that in hPRL like hGH, the free energy comes from interactions between the foot domains and Site 2 serves as a purely regulatory motif.

Human placental lactogen (hPL), another member of the lactogenic hormone family, shares over 80% amino acid sequence homology with hGH.

However it can only bind to the prolactin receptor and not the growth hormone receptor. Mutation of five residues in hPL (located in the putative Site 1) can induce hPL to bind to the growth hormone receptor but 1000-fold weaker to the prolactin receptor (112). It would be interesting to understand the details of hPL binding to the prolactin receptor and determine if the mechanism is similar to hGH or hPRL or an independent mechanism altogether. Do all the lactogenic hormones share a similar “induced fit” mechanism of receptor binding? The lactogenic hormones belong to a larger family of cytokines such as IL-6 (PDB# 1ALU), erythropoietin (PDB# 1BUY), leptin (PDB# 1AX8), oncostatin M (PDB# 1EVS) and others that share a similar three-dimensional structure. All these hormones are of pharmaceutical significance and understanding the mechanism of receptor binding by all these molecules could result in the development of a new generation of rationally designed therapeutics.

122

A B

Site 1 Site 2 Site 1 Site 2

Figure 7.1 Comparison of the NMR structure (A) and theoretical model (B) of hPRL All the residues shown in figure 6.13 were selected (F37, F40, Y44, F50, Y168, Y169, H173) and the orientation of the functional groups compared between the theoretical model and the published solution structure (PDB# 1N9D)(111).

123

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