Sites and Roles of Arrestin Phosphorylation in Regulating Interactions between Arrestin and Rhodopsin of Squid Eyes
Xinyu Guan
A thesis submitted in conformity with the requirements for the degree of M.Sc. Graduate Department of Pharmacology and Toxicology University of Toronto
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Xinyu Guan
A thesis submitted in conformity with the requirements for the degree of M.Sc. Graduate Department of Pharmacology and Toxicology University of Toronto, 2008
Abstract
In squid (Loligo pealei], activation of phototransduction is mediated by rhodopsin isomerization to metarhodopsin and the activation of a phospholipase C pathway, resulting in depolarization of the retinal membrane. Inactivation is mediated by squid rhodopsin kinase (SQRK) and arrestin. Arrestin binding to metarhodopsin is the key inactivation step and SQRK-mediated phosphorylation of rhodopsin is not required for arrestin binding.
SQRK can also phosphorylate arrestin, a function that is not shared by other receptor kinases.
To determine the site(s) and function of arrestin phosphorylation, I expressed recombinant arrestins in E. coli and used site-directed mutagenesis. Squid arrestin was phosphorylated at Ser392 and/or Ser397 by SQRK. Arrestin phosphorylation did not affect arrestin binding to rhodopsin in the light but inhibited its binding to dark-adapted membranes. Furthermore, the phosphorylation significantly increased arrestin dissociation from dark-adapted membranes at high salt concentrations in vitro. This work suggested that the role of arrestin phosphorylation was to increase dissociation of the protein from rhodopsin once it has been photoconverted back to inactive state.
ii Acknowledgements
I was born in a small town in China, a place so remote and peaceful that even the keenest journalist may have forgotten its existence. In my world, imagination is the king.
But even in my wildest dreams, I have never anticipated to study abroad or to learn any stories happening inside the world of cells.
Everything happening now seems so unbelievable, but in the same time, is so believable because of you, my mentors Drs. Jane Mitchell and Hee-Won Park. Thank you so much for taking me as your student and for giving your guidance, encouragement and patience throughout my work. I would also like to thank my advisor Dr. James Wells for his advice during the course of my degree. To the additional members of my committee, Drs, J.
Peter McPherson and Scott Heximer, thank you very much for your contribution to my defense. I also want to give my thanks to my colleagues Walter Swardfager, Lick Lai, Lynle
Go, Rosalia Yoon, Shara Hong and Mike Mattocks. It has been my pleasure to learn and work with you.
To you, the student who is about to continue on this work, I wish you read this part before jumping into the discussion section. When I look back the two years I have spent working on this project with Dr. Mitchell, I felt it had been one of the best decisions I had made. "Some are born great, some achieve greatness, and some have the greatness thrust upon them." Unfortunately, you may not register your name in the short list of great people even with great results from this work, but I hope you would one day realize that this may be the first step towards the rest of a meaningful life. Enjoy.
iii Table of Contents
Abstract ii Acknowledgements Hi Table of Contents iv List of Figures vi List of Tables vi List of Abbreviations vii
1. Introduction 1-29 1.1 Invertebrate Eyes 2 1.2 Molecular Pathways of Phototransduction 2 1.2.1 Squid Rhodopsin 5 1.2.2 G Protein 10 1.2.3 Phospholipase C 13 1.2.4 IP3/DAG Signaling Pathway 14 1.3 Termination of Phototransduction 15 1.3.1 Rhodopsin Kinase 16 1.3.1.1 Mammalian G-protein Coupled Receptor Kinase 16 1.3.1.2 Invertebrate Rhodopsin Kinase 17 1.3.2 Arrestin 18 1.3.2.1 Arrestin Structure 19 1.3.2.2 Invertebrate Arrestin 21 1.4 Phosphorylation of Arrestin 27 1.5 Rationale, Research Goals and Hypotheses 28
2. Materials and Methods 29-34 2.1 Materials 29 2.2 Preparation of Dark-Adapted Salt-Washed Rhabdomeric Membranes 29 2.3 cDNA Preparation and Mutation 30 2.4 Production of Recombinant Arrestin 31 2.5 Enrichment of Recombinant Arrestin 31 2.6 Arrestin Phosphorylation Assays 32 2.7 Membrane Association Assays 32 2.8 Membrane Dissociation Assays 33 2.9 Other Methods 33
3. Results 34-58 3.1 Solubility of Recombinant sArr 34 3.2 Recombinant sArr Production and Enrichment 41 3.3 Phosphorylation of Recombinant sArr 45 3.3.1 SQRK-Dependent sArr Phosphorylation 45 3.3.2 Membrane and Calcium Effects on sArr Phosphorylation 47 3.4 Determination of the Sites of Phosphorylation on sArr 47 3.5 Membrane Binding Assays 50 3.6 Membrane Dissociation Assays 54
iv 4. Discussion 59-71 4.1 Recombinant sArr Production and Purification 59 4.2 Phosphorylation of sArr by SQRK 63 4.2.1 Membrane Effect on sArr Phosphorylation 63 4.2.2 Calcium Effect on sArr Phosphorylation 65 4.2.3 Phosphorylation and Clathrin-Binding Sites on sArr 66 4.3 Functional Role of the Phosphorylation 67 4.3.1 Phosphorylation and Arrestin Binding to Membranes 67 4.3.2 Phosphorylation and Arrestin Dissociation from Membranes 68 4.4 Modeling the Arrestin-Mediated Receptor Desensitization and Future Studies 69
5. Appendix 72
6. Reference 73-81
v List of Figures
Introduction Figure 1. Squid Eye and Camera-Type Vision 3 Figure 2. Squid Photoreceptors 4 Figure 3. Activation of Invertebrate Visual Signaling Pathway 6 Figure 4. Three-Dimentional Structure of Squid Rhodopsin 8 Figure 5. Sequence Alignment of Loligo pealei and Todarodes pacificus Rhodopsins 9 Figure 6. Hypothetical Structure of a Complex Composed of GtaPy with Rhodopsin 12 Figure 7. Three-Dimensional Structure of Mammalian B-Arrestin 20 Figure 8. Model of Mammalian Arrestin Binding to Activated Receptors 22 Figure 9. Alignment of Amino Acid Sequences of Arrestins 25
Results Figure 10. Solubility of rsArrWT & IPTG Concentrations and Growth Temperatures 36 Figure 11. Solubility of rsArrWT in Origami Cells 38 Figure 12. Solubility of GST- and HisMBP-Tagged rsArrWT 39 Figure 13. Effects of Lysis Buffer pH on rsArrWT Solubility 40 Figure 14. Representation of the rsArrWT Enrichment Process 42 Figure 15. Representation of the rsArr3A Enrichment Process 43 Figure 16. Representation of the rsArr2A3 Enrichment Process 44 Figure 17. SQRK-Dependent sArrWT Phosphorylation 46 Figure 18. Membrane Effects on rsArrWT Phosphorylation 48 Figure 19. Calcium Effect on rsArrWT Phosphorylation 49 Figure 20. Phosphorylation of rsArrWT and rsArr3A 51 Figure 21. Phosphorylation of rsArrWT, rsArr3A and rsArr2A3 52 Figure 22. Light-Dependent Membrane Binding of rsArrWT and rsArr3A 53 Figure 23. Effects of Phosphorylation on nsArr Binding to Membranes 55 Figure 24. Comparison between phosphorylated and unphosphorylated nsArr 56 Figure 25. Dissociation of nsArr from Dark Adapted Membranes 58
Discussion Figure 26. Model of Rhodopsin Cycle in Invertebrate Photoreceptors 71
List of Tables
Table 1. Estimation of arrestin concentration and yield per 120 g cell paste 45
VI List of Abbreviation
Xmax: maximum absorbance wavelength AEBSF: 4-(2-Aminoethyl] benzenesulfonyl fluoride hydrochloride ATP: adenosine triphosphate C-terminus: carboxyl-terminus cDNA: complementary dioxyribonucleotide sequence cGMP: cyclic guanosine monophosphate DAG: diacylglycerol DTT: dithiothreitol E. coli: Escherichia coli EDTA: ethylenediaminetetraacetic acid EGTA: ethylene glycol tetraacetic acid G protein: guanine nucleotide-binding protein GBy: (3- and y-subunits of guanine nucleotide-binding protein GDP: Guanosine diphosphate Gt> mammalian visual guanine nucleotide-binding protein, transducin GTP: Guanosine triphosphate GTPyS: Guanosine 5'-(3-0-thio)triphosphate GPCR: Guanine nucleotide-binding protein-coupled receptors HEK293: human embryonic kidney cell line IPTG: Isopropyl (3-D-l-thiogalactopyranoside IP3: inositol 1,4,5-trisphosphate iGq: invertebrate guanine-nucleotide binding protein, q subclass iGqCc: invertebrate guanine-nucleotide binding protein, q subclass, a subunit inaE gene: Drosophila gene encoding a diacylglycerol lipase LB: Lysogeny Broth mGq: mammalian guanine-nucleotide binding protein, q subclass N-terminus: amino-terminus nsArr: native squid arrestin Ni-NTA: nickel-nitrilotriacetic acid agarose resin
vii OD6oo: optical density at 600 nm wavelength PIP2: phosphatidylinositol 4,5-bisphosphate PH: pleckstrin homology PLC: phospholipase C RGS: regulator of G-protein signaling rsArrWT: wild type recombinant squid arrestin rsArr3A: recombinant squid arrestin with triple mutation (S392A, T396A and S397A) rsArr2Al: recombinant squid arrestin with double mutation (S392A and T396A) rsArr2A2: recombinant squid arrestin with double mutation (T396A and S397A) rsArr2A3: recombinant squid arrestin with double mutation (S392A and S397A) Retinal: retinaldehyde rdgA gene: Drosophila gene encoding a diacylglycerol kinase rdgC gene: Drosophila gene encoding a Ca2+-dependent phosphatase SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis sArr: squid arrestin SQRK: squid rhodopsin kinase TB: Terrific Broth TRP channel: transient receptor potential channel TRPL channel: TRP-like channel
viii 1. Introduction
Guanine nudeotide-binding protein (G protein)-coupled receptors (GPCRs)
constitute by far the largest family of membrane receptors. There are more than 800
known GPCRs in the human genome, and they are involved in the regulation of nearly all
physiological functions [Premont and Gainetdinov 2007]. Various ligands have been
identified for GPCRs, which include peptides, lipids, amino acids, and small molecules such
as catecholamine, acetylcholine, and even photons. Since GPCRs play such a critical role in
human health, understanding how they are regulated is of great importance to physiology,
pharmacology and medicine.
Opsins are members of the largest subfamily of GPCRs, constituting —90 % of all
GPCRs. Their function in phototransduction has been established in many species
(Langmack and Saibil 1991; Palczewski and Saari 1997; Purcell and Crosson 2008). In
mammalian eyes, the opsin-activated signaling pathway is mediated by inhibition of the
second messenger - cyclic guanosine monophosphate (cGMP). However, this pathway is
not commonly utilized in non-visual cells (Lott, Wilde et al. 1999). In contrast, invertebrate
opsin stimulates inositol 1,4,5-trisphosphate (IP3) production during phototransduction, a
mechanism also adapted by many GPCRs such as ai-adrenergic and angiotensin receptors
in regulating the cardiovascular system (Lefkowitz 2007) and some of the muscarinic
acetylcholine receptors in facilitating functions of the central and peripheral nervous
systems (Wess, Eglen et al. 2007). These aspects of invertebrate opsin-mediated signal transduction make it a valuable model for studying the signaling pathways that utilize IP3
as second messengers. In this thesis, I will highlight recent understandings of how
invertebrate rhodopsin-mediated signaling is regulated and terminated, especially in the
squid {Loligo pealei) visual system.
1 1.1 Invertebrate Eyes
Of a total of 8 types of eyes in nature, cephalopod eyes have been shown to most
closely resemble their human counterparts in regards to both the optical process and
morphological structures (Land and Nilsson 2002). Both eyes exhibit camera-type
features, consisting of a cornea, an anterior chamber, an iris, a lens, and a retina arranged
in a distal to proximal order (Walrond and Szuts 1992) (Figure 1). Light refracted through the lens forms an inverted image on the retina. The retina is composed of layers of
photoreceptor cells - the functional units that convert photon energy to neuronal signals
that are relayed further to the brain.
Unlike most mammals which have both rod and cone photoreceptors, the squid has
only one type of photoreceptor (Arendt 2003). These cells are bilobular in morphology and consist of a distal rhabdomeric lobe and a proximal arhabdomeric lobe (Figure 2). The rhabdomeric surface is packed with microvilli which hold visual pigments in their membranes. This microvillar organization maximizes surface area for capturing light photons (Cohen 1973). Underneath the microvilli, an extensive network of endoplasmic reticula called submicrovillar cisternae function as intracellular compartments for calcium storage (Walrond and Szuts 1992). The arhabdomeric lobe contains cell nuclei and other essential organelles for survival, and gives rise to axons at its proximal end (Caiman and
Chamberlain 1982).
1.2 Molecular Pathways of Phototransduction
Light photons reach the eye in the form of energy that can be absorbed by a chromophore known as retinaldehyde (retinal) - a Vitamin A analogue. The retinal is embedded in the membrane protein - opsin. Collectively, the complex is referred to as rhodopsin or the visual pigment. Energy absorbed by retinal activates rhodopsin, which is
2 Optic nerve
Figure 1. Squid Eye and Camera-Type Vision. A photograph shows a squid eye which contains no eyelids and controls the amount of incoming light by opening and closing its pupil (A)1. An anatomical representation of a squid eye is illustrated in panel B2. The camera-type vision is represented in panel C3. Inversed images are formed on the retina which contains both rod and cone photoreceptors in humans whereas squid has only one type of photoreceptors.
1 Reproduced from http://www.mnh.si.edu/natural_partners/squid4/ArchiteuthisAdaptations.htrnl Search for Giant Squid, Smithsonian National Museum of Natural History. 1999 2 Reproduced from Strickberger, M. W. 1990. Evolution. Jones and Bartlett Publishers, Boston, MA. 3 Reproduced from http://www.lhup.edu/~dsimanek/scenario/miscon.htm Didaktikogenic Physics Misconceptions, Lock Haven University of Pennsylvania.
3 Figure 2. Squid Photoreceptors. A single squid photoreceptor is shown in panel A4. The arrow heads point to the distal rhabdomeric lobe, and the arrows point to the proximal arhabdomeric lobe. Schematic representation of a squid photoreceptor is shown in panel B5. The retina (center] is formed by a layer of photoreceptors. The tip of the rhabdomeric lobe points directly to the incoming light. The microvilli of rhabdomeric membranes (right) are packed with photoreceptors in orthogonal directions, which not only maximize light capturing but also confer sensitivity to polarized lights (Cohen 1973).
4 Figure adapted from Walrond etal. J Neurosci 12:1490-501. (1992).
5 Reproduced from http://people.crystbbk.ac.uk/~ubcgl6z/old/squid.html. Squid photoreceptors, by Helen Saibil and Tony Davies (University College London). Accessed on July 12, 2008.
4 accompanied by rotation of its double bond between the 11th and 12th carbons to change conformation from 11-c/s to all-trans retinal (Chang 2001). This isomerization may subsequently alter rhodopsin's overall conformation, resulting in intracellular responses.
Molecular pathways of the intracellular response have been well characterized in the mammalian vision. Photoexcited mammalian rhodopsin interacts with a heterotrimeric guanine nucleotide-binding protein (G protein) of the Gi family known as Gt or transducin, which in turn activates cGMP phosphodiesterase that breaks down cGMP. The decrease in intracellular cGMP level will then lead to closing of cGMP-gated membrane cation channels and transient hyperpolarization of photoreceptor cells (Liebman, Parker et al. 1987).
Biochemical and mutagenesis studies have provided valuable insights into the molecular pathway of invertebrate phototransduction. Instead of Gt, invertebrate rhodopsin is coupled to invertebrate Gq protein (iGq). Activation of iGq by photoactivated rhodopsin leads to activation of an effector protein - phospholipase C (PLC) (Yoshioka,
Inoue et al. 1985; Mitchell, Gutierrez et al. 1995). The activated PLC then hydrolyzes a membrane-bound inositol lipid precursor - phosphatidylinositol 4,5-bisphosphate (PIP2), releasing second messengers - IP3 and diacylglycerol (DAG) (Fein, Payne et al. 1984;
Blenau and Baumann 2001) (Figure 3). These messengers induce both Ca2+ release from submicrovillar cisternae and opening of cation channels in the plasma membrane, together resulting in a significant increase in intracellular Ca2+ level and transient depolarization of photoreceptor cells (Hardie and Minke 1993; Hardie and Minke 1995).
1.2.1 Squid Rhodopsin
Squid rhodopsin is also the main protein component in rhabdomeric microvillar membranes of photoreceptor cells. It shares conserved residues of all GPCRs and 75 % homology with octopus rhodopsin and 35 % homology with mammalian rhodopsin
5 Submicrovillar Cisternae
Figure 31. Activation of Invertebrate Visual Signaling Pathway. As illustrated in this figure, in invertebrate photoreceptors, light activated rhodopsin subsequently activates iGq. In turn, activated iGq stimulates effector protein PLC that hydrolyzes membrane-bound PIP2 to produce both IP3 and DAG as secondary messengers. IP3 induces IP3 receptor (IP3-R)-gated submicrovillar cisternae to release Ca2+, whereas DAG may activate membrane cation channels (TRP and TRPL channels) to initiate Ca2+ influx. The end result for the rise of intracellular Ca2+ level will be transient cell membrane depolarization, leading to signal relay from photoreceptors to neurons.
1 Figure adapted from Blenau et al. Arch Insect Biochem Physiol 48(1): 13-38. (2001).
6 (Davies, Gowen et al. 2001). Like other GPCRs, squid rhodopsin has seven transmembrane a-helices and a retinal molecule embedded in its hydrophobic core. A protonated Schiff base covalently links the retinal molecule and the £-amino group of Lys305 on helix VII of squid opsin (Go and Mitchell 2004; Prete 2004). Variations in the retinal binding site in different species are considered one of the key factors determining the maximum absorbance wavelength (A,max) of rhodopsin. Squid rhodopsin, for example, has a kmax of
493 nm for blue-green light (blue: 450-495 nm; green: 495-570 nm) (Hubbard 1956;
Bruno and Svoronos 2005).
Early investigations of the molecular structure of invertebrate rhodopsin have been extended to species such as Drosophila (Zuker, Cowman et al. 1985) and cephalopods
(Ovchinnikov Yu, Abdulaev et al. 1988) including squid (Davies, Gowen et al. 2001). These studies contributed valuable information to the primary sequence determination.
Recently, the crystal structure of squid (Todarodes pacificus) rhodopsin has been solved
(Murakami and Kouyama 2008) (Figure 4). Todarodes pacificus rhodopsin contains 448 amino acids that share 83 % homology with the primary sequence of Loligo pealei rhodopsin (Go and Mitchell 2003). In comparison to their mammalian counterparts, both pacificus and pealei rhodopsins are about 100 amino acids longer. The extra peptides form a carboxyl terminal (C-terminal) extension containing a proline-rich region (Go and
Mitchell 2004; Murakami and Kouyama 2008) (Figure 5). This proline-rich region is constituted by 9-10 repeats of a pentapeptide: Pro-Pro-Gln-Gly-Tyr, and so far has only been found in the cephalopod rhodopsin (Venien-Bryan, Davies et al. 1995). Functional impact of the proline-rich region is not clear; however, it may facilitate nonspecific protein-protein interactions by potentially serving as a binding site (Williamson 1994). In the case of squid rhodopsin, the proline-rich region may be also of importance to maintain
7 C3 Loop
Figure 46. Three-Dimentional Structure of Squid Rhodopsin (brown) and Bovine Rhodpsin (cyan). The squid (Todarodes paciflcus) rhodopsin (Protein Data Bank [PDB] ID: 2Z73) is superpoimposed with bovine rod rhodopsin (PDB ID: 1U19). In addition to the 7 transmembrane a-helices (I to VII], the squid rhodopsin contains 2 extra helices labeled as helices VIII and IX, and both are close to the protein's C-terminus. Overall, the bovine and squid rhodopsins are suprisingly similar in structure, given that their amino acid sequence homology is only 29 %. However, the most notable difference between the two is the third cytoplasmic (C3) loop that is formed between the V and VI a-helices. In squid rhdopsin, the C3 loop extends largely into the cytosol, whereas its mammalian conterpart is mostly confined in the membrane region.
6 Figure taken from Murakami etal. Nature 453: 363-7. (2008).
8 : Pealei l no: i. ..:.•.•:.•;;•;•::: •::•:.•: •:.•.:.•.•,- ,'." [AAVYYSLGIFIGICGIIGCVGNGIVIYLFT 60
Pacificus l ::';P:"'\VN"":w;r-!i .-••;•-;••k-k-k-k kk ' v±k. • • : .;.v. [DAVYYSLGIFIGICGIIGCGGNGIVIYLFTMJ k T 60
Pealei 61 K]TKSLQT[PANMFIINLAFSDFTFSLVNGFPLMTISCFL]KYWVFG[NAACKVYGLIGGIFGL 120 Pacificus 61 K]TKSLQT[PANMFIINLAFSDFTFSLVNGFPLMTISCFL]KKWIFG[FAACKVYGFIGGIFGF 120 ICl TM2 *ECfc * * TM3 k
Pealei 121 MSIMTMTMISIDRYNVIG]RPMSASKKMS[HRKAFIMIIFVWIWSTTWAIGPIF]GWGAYSLE 180 Pacificus 121 MSIMTMAMISIDRYNVIG]RPMAASKKMS[HRRAFIMIIFVWLWSVLWAIGPIF]GWGAYTLE 180 * * IC2 * TM4 * * * *
Pealei 181 GVLCNCSFDYISRD[SSTRSNIVCMYLFAFMCPIIVIFFCYFNIVMSVANHXKEMAAMAKR] 240 Pacificus 181 GVLCNCSFDYISRD[STTRSNILCMFILGFFGPILIIFFCYFNIVMSVSNHEKEMAAMAKR] 240 EC2 * * k*kk kk k* TMS * k 1C3
Pealei 241 LNG[KELRRAQAGASAEMKLGKISVVIVTQFLLSGSPYAMVALLAQF]GPLEWVT[RYAAQLP 300 Pacificus 241 LNA[KELRKAQAGANAEMRLAKISIVIVSQFLLSWSPYAVVALLAQF]GPLEWVT[PYAAQLP 300 k k k k *TM6 -k k -k -k EC3 *
Pealei 301 VMFAKJobASAIHNPMIYSV]SHPKFREAIASNFPWILTCC337QKDEKEIEDEKDAEAEIPACEQS360 Pacificus 301 VMFAK305ASAIHNPMIYSV]SHPKFREAISQTFPWVLTCC337QFDDKETEDDKDAETEIPAGESS360 •k -k k -k
Pealei 361 -. jj..:t-..i.-J-.^:'.'. •'] ..-.[-:.• „/:-:,:., ..J J. v.. -- ii. :_;_, /-/_!.-• '-^-.i J 2'f. •-LL-.":'!! .- 415 Pacificus 361 i- 1---v.:.-...•.;•'.ii-::'••.••:'•;;•:••.„'.v;. ...,._••.._ ...>'»; :.\^ V.TO.i^K-nr'-jv/rj^T 419 *• * * * * -k -k-k-k-k -k -k
Pealei 416 ..... 442 Pacificus 420 . ... 448
Figure 5. Sequence Alignment of Loligo pealei and Todarodes pacificus Rhodopsins. Full length Loligo pealei (EMBL ID: Q6SSJ0] and Todarodes pacificus (EMBL ID: P31356) rhodopsin amino acid sequences are aligned. The sequence homology between the two is 83 %. Seven transmembrane helices (TM) are illustrated in brackets. The N- and C-termini are highlighted in grey. Three intracellular loops (IC), three extracellular loops (EC], and the proline-rich C-terminus are underlined. Retinal binding site Lys305, rhodopsin palmitoylation site Cys337, and potential rhodopsin phosphorylation site Ser360 are all bolded. The dots indicate conserved amino acids, and asterisks indicate substituted amino acids. The dashes in between amino acids denote gaps that are introduced for optimal alignment. rhodopsin's mobility in membranes. When reconstituted with squid photoreceptor membranes, the truncated rhodopsins tend to form rigid and organized crystal lattices in the membrane (Venien-Bryan, Davies et al. 1995]. This observation may also explain that the C-terminal truncated rhodopsin is prevalently used in crystallography studies.
Upon light exposure, mammalian rhodopsin undergoes a cyclic decomposition and regeneration. In its activated state, the mammalian rhodopsin, also referred to as metarhodopsin II, dissociates from all-trans retinal by hydrolysis of the Schiff base [Becker
1988). The resulting opsin does not respond to light until re-associated with 11-c/s retinal.
This process of mammalian rhodopsin losing retinal and photosensitivity is known as chromophore bleaching. Regeneration of rhodopsin requires both enzymatic conversion of all-trans retinal to 11-c/s retinal and association of the latter with opsins (Pepe 2001).
On the hand, invertebrate rhodopsin undergoes a different process of regeneration after light activation. The activated rhodopsin, known as metarhodopsin, remains bound to all-trans retinal (Davies, Gowen et al. 2001). Furthermore, invertebrate rhodopsin and metarhodopsin are photoconvertible at specific wavelengths (Pepe 2001). For example, the squid rhodopsin that is activated at 493 nm can absorb light again, as metarhodopsin, at 500 nm to allow conversion back to rhodopsin (Naito, Nashima-Hayama et al. 1981). It has also been demonstrated that cephalopod metarhodopsin can also isomerize back to rhodopsin in the dark in a prolong period of time (Naito, Nashima-Hayama et al. 1981).
1.2.2 G Protein
There are 16 heterotrimeric G protein subtypes identified in mammals, which are functionally grouped into four classes: Gs, Gi, Gq, and G12 (Dromey and Pfleger 2008).
Invertebrate visual G proteins (iGq) are homologous to the mammalian Gq. iGq has been identified and characterized in several species including Drosophila (Bloomquist, Shortridge et al. 1988; Running Deer, Hurley et al. 1995), Limulus (Fein and Corson 1981;
Kirkwood, Weiner et al. 1989) and squid (Pottinger, Ryba et al. 1991; Go and Mitchell
2007). It is composed of three subunits (a, (3, and y) with the oc-subunit (iGqa) serving as the main functional unit in activating the effector protein - PLC. Although there is evidence suggesting that the mammalian By subunits may also play a regulatory role in stimulating PLC (Katz, Wu et al. 1992; Berridge 1993; Running Deer, Hurley et al. 1995), the invertebrate By has no effect on PLC activation in squid visual system (Mitchell,
Gutierrez et al. 1995).
The full-length iGqa cDNA consists of 1065 nucleotides (GenBank accession number: AF521583) and encodes for a protein sharing 77 % sequence homology with the a-subunit of mammalian Gq proteins (mGqa) (Go and Mitchell 2007). The 3-dimentional structure of the mammalian Gq has been solved, with a chimeric G;/qa (N-terminal helix substitution of Gqa by da) in complex with GBiy2 (Figure 6) (Tesmer, Kawano et al. 2005).
All members of the G-protein family share two highly conserved domains - GTPase and helical domains (Kikkawa, Tominaga et al. 1996; Dorlochter, Klemeit et al. 1997; Go and
Mitchell 2003). The GTPase domain hydrolyses bound GTP to yield GDP in its nucleotide- binding pocket, which terminates a-subunit's activity. Functional roles of the helical domain is not clear, but structural data suggest that they form a "lid" over the nucleotide- binding pocket, burying the bound GTP or GDP (Lambright, Sondek et al. 1996).
Three sites on mGqa have been identified to regulate interactions with receptors, including the N-terminus, a linker region between the GTPase domain and helical domain, and the C-terminus (Itoh, Cai et al. 2001) (Figure 6). iGqa shares significant sequence homology with mGqa in all three sites; however, at the N-terminus, iGqa lacks a six-amino acid extension - MTLESI that is found on mGqa (Go and Mitchell 2007). Our previous data
11 Rhodopsin
r- r> > v, >J ;j S» i) Y) J t Ci U u '-.( \i (i Ki U. U i, '1 % , % v. "\ -,•••, v. •. > ••,-., Vi ') * i *s J {•\ U •.< ;\' *c" i( it •,( a: c /, .-7 // ft ?t )t t) •'/ ,«, -••' - ,i ?:• I* ••'/ /" .// ti )/ t) >/ ? /.* // r/ /•• i/ // // '/ tt •>•' <
GTPase domain-
Helical domain
7 Figure 6 . Hypothetical Structure of a Complex Composed of Gtapy with Rhodopsin. The schematic representation shows that mammalian rhodopsin (PDB ID: 1GZM) interacts with Gt a-subunit (PDB ID: 1G0T) at three potential sites: the N- and C-termini and the linker region between GTPase [blue] and helical (purple) domains. The Ser240, on the third intracellular loop of rhodopsin, was proposed to interact specifically with G-proteins (Itoh, Cai et al. 2001). The G(3 and Gy subunits are labeled in green and gold, respectively. GTP, which is embedded in the Gcc subunit, is highlighted in red. The retinal linked to rhodopsin is in yellow.
7 Figure taken from Oldham et al Nat Rev Mol Cell Biol 9: 60-71. (2008).
12 suggested that the MTLESI extension might hinder the interaction between activated receptors and Gq a-subunit, which was demonstrated by the lower efficacy of iGqa in the downstream PLC activation. When stimulated through muscarinic acetylcholine receptor-
1 or oc-adrenergic receptors in HEK cells, mGqa-mediated PLC activation was significantly less than the iGqa-mediated PLC activation, as measured by the amount of IP3 produced
(Go and Mitchell 2007). However, either removing the extension or stimulating mGqoc directly with AIF4" achieved almost equivalent PLC activation to that of iGqoc, suggesting that the N-terminus region of iGqoc plays an important role in interacting with receptors
(Go and Mitchell 2007).
1.2.3 Phospholipase C
The invertebrate phospholipase C (PLC) has been identified in various species including Loligo pealei (Mitchell, Gutierrez et al. 1995), Drosophila (Yoshioka, Inoue et al
1985; Shortridge, Yoon et al 1991), and Limulus (Fein, Payne et al. 1984). The squid visual
PLC is 140 kDa in size and consists of six distinct modular domains: a pleckstrin homology
(PH) domain, two PLC catalytic domains (X and Y), a calcium binding domain, and P- and
G-box motifs (Mitchell, Gutierrez et al. 1995; Mayeenuddin, Bamsey et al. 2001). The PH domain is widely found in proteins with membrane binding activities. N-terminus of the
PH domain binds to phosphoinositide head group of membrane lipids in a stereo-specific manner (Lemmon, Ferguson et al. 1995). G-protein interacts and activates PLC possibly through the P- and G-box motifs at the C-terminal region (Wu, Katz et al. 1992; Lee, Shin et al. 1993).
PLC, when activated by iGqa, hydrolyzes membrane-bound PIP2 to produce second messengers IP3 and DAG. Aside from its function as an effector protein, our previous assays demonstrated that squid visual PLC stimulated the GTPase activity of iGqa by around 5 folds (Mayeenuddin, Bamsey et al. 2001). This observation demonstrates inhibitory effects of squid PLC on iGqcc function, which may further suggest a negative feedback mechanism in regulating iGqa's activity [Mayeenuddin, Bamsey et al. 2001).
1.2.4 IP3/DAG Signaling Pathway
The invertebrate IP3 signaling pathway has been characterized in Drosophila
(Yoshikawa, Tanimura et al. 1992; Montell 1999), Limulus (Brown, Rubin et al. 1984; Fein,
Payne et al. 1984) and Loligo pealei (Wood, Szuts et al. 1989). In response to external stimuli, both IP3 and DAG are formed. IP3 is released into the cytosol to induce calcium release from internal storage gated by IP3 receptors, whereas DAG remains anchored in the cell membrane and may activate cation channels (Hardie 2004). The end result is a significant rise of intracellular Ca2+ level and transient photoreceptor depolarization.
With cloning and characterization of two Drosophila membrane channels - transient receptor potential (TRP) (Montell and Rubin 1989) and TRP-like (TRPL) channels (Phillips, Bull et al. 1992), it is now known that the rise of intracellular Ca2+ is mediated partially by influx of cations through these two channels (Niemeyer, Suzuki et al.
1996). However, the intrinsic link between production of IP3/DAG and opening of TRP and
TRPL channels remains elusive. Recent studies suggested that the DAG and/or its metabolites played a key role in activating the membrane channels (Chyb, Raghu et al.
1999; Raghu, Usher et al. 2000; Kwon and Montell 2006). It was first tested by a whole-cell recording study with wild type Drosophila photoreceptors by Chyb et al. The authors demonstrated that linolenic acid reversibly activated TRPL channels in a dose-dependent manner (Chyb, Raghu et al. 1999). Furthermore, mutations in the rdgA gene in Drosophila, which encodes for DAG kinase, resulted in a constitutive activation of TRP and TRPL channels (Raghu, Usher et al. 2000). Since the DAG kinase is primarily responsible for regenerating PIP2 from DAG, it is plausible that inactivation of this enzyme will lead to accumulation of DAG in the membrane, which may provide constitutive stimuli to the membrane channels (Hardie, Martin et al. 2002). A recent study by Leung etal. identified the inaE gene in Drosophila, which encodes for another DAG metabolizer - DAG lipase
(Leung, Tseng-Crank et al. 2008). The DAG lipase converts DAG to 2-monoacylglycerol - a precursor of poly-unsaturated fatty acids. Leung et al. further demonstrated that the mutation in inaE gene significantly decreased the amplitude of light responses in photoreceptors, indicating that the poly-unsaturated fatty acids may be key factor activating TPR and/or TPRL channels (Leung, Tseng-Crank et al. 2008).
1.3 Termination of Phototransduction
The termination of signal transduction is essential for maintenance of the sensitivity and dynamics of cells in response to external stimuli. It is of particular importance in phototransduction systems, since the lifetime of activated rhodopsin is far longer than the required time for resolution (Vishnivetskiy, Paz et al. 1999). One termination process, known as receptor desensitization, has been studied extensively with bovine rhodopsin and mammalian (3-adrenergic receptors (Ferguson, Downey et al. 1996;
Palczewski 1997; DeWire, Ahn et al. 2007). In both cases, the desensitization occurs in two steps. The first step involves phosphorylation of intracellular serines and/or threonines on activated receptors by a specific receptor kinase. The phosphorylation results in limited receptor desensitization, but increases the its binding affinity for another protein called arrestin (Maeda, Imanishi et al. 2003). Arrestin is a soluble cytosolic protein that quenches signal transduction by competing with G proteins for the binding site on activated receptors. The binding of arrestin to receptors terminates signal transduction despite continued presence of stimuli. In the squid visual system, both rhodopsin kinase and arrestin have been identified in our lab, and named as squid rhodopsin kinase (SQRK) and squid arrestin (sArr)
(Mayeenuddin and Mitchell 2001; Mayeenuddin and Mitchell 2003; Swardfager and
Mitchell 2007]. Their functions in regulating and interacting with squid rhodopsin will be discussed as follows.
1.3.1 Rhodopsin Kinase
1.3.1.1 Mammalian G-protein Coupled Receptor Kinase
G-protein coupled receptor kinases (GRKs) are a subgroup of the very large family of serine/threonine kinases. There are seven GRKs in vertebrates, named GRK1 through
GRK7. The GRKs are functionally divided into three classes: GRKl-like, GRK2-like, and
GRK4-like classes. GRK1 (rhodopsin kinase) and GRK7 (photopsin kinase) belong to the
GRKl-like class, which are primarily found in the retina and regulate rhodopsin and photopsin phosphorylation, respectively (Mendez, Burns etal. 2000). GRK2 and related
GRK3 are widely expressed in tissues and share a C-terminal pleckstrin homology (PH) domain (Sterne-Marr, Dhami et al. 2004). The GRK4-like class consists of GRK 4, 5, and 6.
The expression of GRK4 is confined to the testes, whereas GRK5 and GRK6 are widely expressed in the body (Gainetdinov, Premont et al. 2000). All GRKs share a serine/threonine kinase catalytic domain flanked by a conserved N-terminal domain - homologous to the Regulator of G-protein Signaling (RGS) proteins, and a variable C- terminal domain (Singh, Wang et al. 2008). It has been reported that the N-terminal domain may be involved in receptor recognition, while the C-terminal domain functions as sites for post-translational modifications and protein-protein interactions (Palczewski
1997). For an activated GPCR to be phosphorylated by a GRK, the kinase must be recruited to the membrane and form a complex with the receptor. Of the seven types of GRKs, five
(GRKs 1,4, 5, 6, and 7) are post-translationally modified by the addition of fatty acids and are constitutively associated with the membrane (Pitcher, Freedman et al. 1998). In contrast, both GRK2 and GRK3 are soluble in the cytosol and undergo transient recruitment to the membrane after receptor activation (Strasser, Benovic et al. 1986). The
C-terminal PH domains of GRK2 and GRK3 bind to PIP2 in the membrane, facilitating their membrane recruitment (Tesmer, Kawano et al. 2005). GRKs phosphorylate activated receptors at specific sites on C-terminus. There are seven putative phosphorylation sites on the C-terminus of mammalian rhodopsin, consisting of three serines and four threonines (Hargrave 2001). In vitro studies have shown that bovine rhodopsin is phosphorylated at multiple sites - Ser334, Ser338 and Ser343, at the receptor's C-terminus
(Ohguro, Palczewski et al. 1993). An in vivo study confirmed the phosphorylation of multiple sites on rhodopsin in rod cells of living mice (Kennedy, Lee et al. 2001). The functional role of the receptor phosphorylation will be discussed in later sections.
1.3.1.2 Invertebrate Rhodopsin Kinase
Invertebrate receptor kinases have been identified in the visual systems of
Drosophila (dGRKl and dGRK2) (Cassill, Whitney et al. 1991), Limulus (Edwards, Wishart et al. 1989), octopus (octopus rhodopsin kinase) (Kikkawa, Yoshida et al. 1998), and squid
(SQRK) (Mitchell and Mayeenuddin 1998; Mayeenuddin and Mitchell 2001). These kinases are shown to be more homologous to mammalian GRK2 rather than the rhodopsin kinase
- GRK1 (Cassill, Whitney et al. 1991; Mayeenuddin and Mitchell 2001). SQRK also contains the conserved RGS and kinase catalytic domains, and the C-terminal PH domain as found in GRK2 (Mayeenuddin and Mitchell 2001). SQRK phosphorylates squid rhodopsin in a light dependent manner (Mayeenuddin and Mitchell 2001). The site of phosphorylation on squid rhodopsin has yet to be determined, although consensus sites, as being determined with GRK2, are serines or threonines flanked by acidic residues at their amino-terminal side (Onorato, Palczewski et al. 1991). One phosphorylation site was reported on octopus rhodopsin at Ser358 (Ohguro,
Yoshida et al. 1998). This site is homologous to the Ser360 in squid rhodopsin, which is also flanked by an acidic residue (glutamic acid) on its amino-terminal side (Figure 5).
Therefore, it is likely that the Ser360 is the phosphorylation site of squid rhodopsin
(Mayeenuddin and Mitchell 2001). The possibility of having multiple phosphorylation sites as just described in squid rhodopsin may also exist. These sites, however, may be on the upstream of Glu372, since removal of the C-terminus of Todarodes paciflcus rhodopsin at Glu372 did not affect its phosphorylation by rhodopsin kinase (Naito, Nashima-Hayama et al. 1981; Ohguro, Yoshida et al. 1998; Ashida, Matsumoto et al. 2004).
In response to light activation of rhodopsin, SQRK is recruited to the photoreceptor membrane possibly through the interaction of its PH domain with membrane phospholipids (Mayeenuddin and Mitchell 2001). SQRK has also been found to have two
Gy-like regions close to its PH domain, which may enable the kinase to directly interact with squid G(3 (Mayeenuddin and Mitchell 2001).
1.3.2 Arrestin
Arrestin terminates GPCR-medicated signal transduction by inhibiting the interaction between activated receptors and G-proteins. The first member of arrestin family, mammalian rod arrestin, was originally identified as an abundant and soluble protein in rod outer segments of bovine retina (Wacker, Donoso et al. 1977). The rod arrestin is also known as S antigen, a name given because this protein was found to induce autoimmune uveoretinitis, an ocular inflammatory disease (de Kozak, Sakai et al. 1981].
Subsequent to the discovery of rod arrestin, homologues were identified in non-retinal tissues of various species. The latest member of the arrestin family, cone arrestin, was found initially in the pineal gland in rat, which led to the discovery of the arrestin expressed in mammalian cones (Craft, Whitmore et al. 1994). So far, there are four types of mammalian arrestins including two visual arrestins - rod arrestin (arrestin-1) and cone arrestin (arrestin-4), and two non-visual arrestins - B-arrestins 1 and 2 (arrestin-2 and arrestin-3, respectively). The visual arrestins are exclusively expressed in rod and cone photoreceptors, and regulate rod and cone visual pigments. The B-arrestins, however, are widely expressed in the body and regulate most other GPCR functions (Premont and
Gainetdinov 2007).
Both visual and nonvisual arrestins have been identified in invertebrate species including blowfly, Drosophila, Limulus, and squid (Smith, Shieh et al. 1990; Ellis and
Edwards 1994; Mayeenuddin and Mitchell 2003). There are two isoforms of blowfly and
Drosophila arrestins and one isoform of Limulus and squid arrestins.
1.3.2.1 Arrestin Structure
The 3-dimentional structures of both rod arrestin and B-arrestin have been solved
(Hirsch, Schubert et al. 1999; Han, Gurevich et al. 2001) (Figure 7). Overall structures of the two are very similar, despite relatively low homology in their amino acid sequences.
Bovine rod arrestin contains 404 amino acids and consists of an N-domain (residues
8~180), a C-domain (residues 188~362), and a C-terminus (residues 372 to 404). Each domain consists of seven strands of B-sheets and an additional lateral strand (Hirsch,
Schubert et al. 1999). A polar core structure is formed at the interface of the N- and C- domains. In rod arrestin, the polar core is constituted by 5 residues: Asp30, Arg175, and C-Doitiaiii
3BS1 Polar Core Region
Figure 78. Three-Dimensional Structure of Mammalian P-Arrestin. The overall structure of B-arrestin (PDB ID: 1G4M) is saddle shaped. It consists of N- and C-domains and a polar core region at the interface between the two domains. Both N- and C-domains are formed by seven strands of B-sheets, denoted as arrow-shaped ribbons. The C-tail contains a short stretch of B-sheet that runs in parallel with the lateral B-sheet of the N- terminus. The C-tail folds towards the polar core region. There is a short cc-helix [helix I) in the N-domain, which is shown in close proximity to the C-tail. It has been suggested that helix I may interact with the C-tail and facilitate its folding. The recognition site of phosphorylated rhodopsin is within the 158-185 residues in bovine visual arrestin which shares high structural homology with B-arrestins. This site was proposed to be responsible for mammalian arrestins to distinguish phosphorylated and unphosphorylated rhodopsins and to bind to the former with high affinity.
8 Figure taken from Han etal Structure 9: 869-80.(2001). Lys176 of N domain, Asp296, and Asp303 of C-domain, and Arg382 of the C-terminus (Hirsch,
Schubert et al. 1999). These residues are highly conserved in all arrestins (Smith, Shieh et al. 1990; Smith, Greenberg et al. 1995; Mayeenuddin and Mitchell 2003). Their hydrogen- bond interactions are essential in maintaining the protein's structure. The C-terminus contains a short (3 sheet that runs in parallel to the lateral (3-strand of the N-terminus. In mammalian arrestin's inactive state, the C-terminus folds into the protein's core region.
Upon activation, initial interactions between arrestin and charged residues on the C- terminus and intracellular loops of receptors may disrupt the polar core structure (Figure
8). Breakdown of the polar core would increase arrestin's structural flexibility and release the C-terminus for other regulations (Hirsch, Schubert et al. 1999). The increase of flexibility and potential conformation changes may eventually result in the formation of a stable complex between arrestin and activated receptors (Gurevich, Dion et al. 1995;
Vishnivetskiy, Paz et al. 1999). The formation of arrestin-receptor complex, therefore, may effectively hinder G protein interactions with the receptor, resulting in receptor desensitization and the termination of signal transduction.
1.3.2.2 Invertebrate Arrestin
Squid arrestin (sArr) has been cloned previously in our lab (Mayeenuddin and
Mitchell 2001; Mayeenuddin and Mitchell 2003). It is exclusively expressed in the eye. The cDNA of full length sArr contains a 1203-nucIeotide open reading frame that encodes a single strand protein of 400 amino acids (Mayeenuddin and Mitchell 2003). Sequence analysis of sArr protein has shown homology with Limulus arrestin (37%), bovine rod arrestin (37%), and the highest homology with mammalian (3-arrestins (43~44 %)
(Mayeenuddin and Mitchell 2003). Like other arrestins, sArr also contains a polar core structure formed by residues - Asp26, Arg169, Asn294, Asp300, and Arg381 of the N- and C- Receptor Receptor
Active Arrestin
Figure 89. Model of Mammalian Arrestin Binding to Activated Receptors. This model demonstrates a proposed receptor binding mechanism of arrestins. The N- and C- domains are shown in pink and cyan, respectively. The polar core region is represented as ball-stick figures at the interface between the two domains. In the inactive state, the C-tail of arrestin is buried inside the protein's core region. Upon interaction with an activated receptor, the polar core is disrupted, which gives rise to higher domain flexibility and release of the C- tail. The N- and C-domains then form a stable complex with the activated receptor. The helix I [yellow, ball-shaped structures] anchors in the membrane with its hydrophobic side, further stabilizing the arrestin-receptor complex.
9 Figure taken from Han etal Structure 9: 869-80. (2001). domains and C-terminus.
Main functions of sArr have been characterized in our lab. Native sArr (nsArr),
extracted and purified from squid eyes, was shown to inhibit the intrinsic GTPase activity
of iGqa in a dose-dependent manner, when reconstituted with rhobdomeric membranes
(Swardfager and Mitchell 2007). Since the GTPase activity is stimulated by light-activated
rhodopsin, the inhibitory effect of sArr on GTPase activity may be also a measure of
disrupted coupling between rhodopsin and iGqa. In another finding, we demonstrated that sArr bound to the membranes (the main source of rhodopsin) in a light-dependent manner, supporting that the binding of sArr may physically block the interaction between rhodopsin and iGqoc (Swardfager and Mitchell 2007). The light-induced membrane binding was also robust; a 20-minute exposure induced an average of 7.2 fold increase in arrestin binding to membranes in the light compared to that in the dark (Swardfager and Mitchell
2007). Since rhodopsin is the one that confers light sensitivity in membranes, the binding difference between light and dark suggests that sArr is able to discriminate between the light-activated and inactive conformations of squid rhodopsin. After all, we have concluded that sArr binds specifically to light-activated rhodopsin and may sterically prevent the latter from coupling and activating iGqa, resulting in receptor desensitization and termination of phototransduction.
A significant difference between the invertebrate and mammalian phototransduction systems is that invertebrate arrestin binding to metarhodopsin does not require rhodopsin phosphorylation (Bentrop, Plangger et al. 1993; Plangger, Malicki et al. 1994), which is the case for mammalian arrestins (Gurevich and Benovic 1997). By measuring light-dependent binding of GTPyS to G-proteins, Khana et al first demonstrated that the inactivation of squid photoreceptors was independent of ATP and kinases (Kahana, Robinson et al. 1992). In addition, this group also demonstrated that the
inactivation required the presence of soluble proteins, presumably the arrestin as
presently knew. Another group led by Plangger et al. demonstrated that in vitro blowfly arrestin bound to unphosphorylated metarhodopsin with comparable affinity to that of phosphorylated metarhodopsin (Plangger, Malicki et al. 1994). In a reconstitution assay, the group also investigated the time course for arrestin to bind to metarhodopsin and the phosphorylation of metarhodopsin. They showed that the binding of arrestin to metarhodopsin was rapid, below their shortest detectable time of 30 seconds; in contrast, the phosphorylation of metarhodopsin proceeded slowly after arrestin binding, requiring
9 minutes for 50 % metarhodopsin phosphorylation (Plangger, Malicki et al. 1994). This time course was also consistent with previous measurements in the intact blowfly retina
(Paulsen and Bentrop 1983). A more recent study also confirmed that invertebrate arrestin binding was independent of receptor phosphorylation, in which Drosophila Arr2 bound to activated rhodopsin in the absence of phosphorylation (Alloway and Dolph
1999). These findings unequivocally demonstrate that invertebrate arrestin binding to rhodopsin is only determined by the receptor activation but not phosphorylation.
The underlining differences between invertebrate and mammalian arrestins that result in the binding differences to phosphorylated and unphosphorylated metarhodopsins are still unknown. Various groups, however, have suggested that this binding difference may be determined by the phosphorylation recognition region in arrestin (amino acids 158-185 in bovine rod arrestin) (Figure 9A) (Gurevich and Benovic
1993; Plangger, Malicki et al. 1994; Gurevich and Gurevich 2006). Interestingly, invertebrate arrestins of Drosophila, blowfly, Limulus, and squid, have all consistently substituted the Arg171 of bovine rod arrestin with a neutral serine. In addition, Arg182 in Bovine (Rod) N' -158DVEEDKIPKKSSVR171LLIR175KIQHAPR182DMG185-C Squid N' -154MDDKIHKRNSVS165LSIR169KLSYFEF176GSD179-C Drosophila N' -152EDDRQHKRSMVS163LVIK167KLQYAPL174NRG177-C Limulus N' -159EDEKPHKRNSVS170MAIR174KLQYAKP181SPL184-C'
Figure 9A. Alignment of the Phosphorylation Recognition Domain of Arrestins. As shown above, the recognition region contains multiple charged amino acids [bold], and most of these are conserved across different arrestins. However, the positively charged Arg171 and Arg182 in bovine arrestin are substituted by neutral amino acids (red] in all the invertebrate arrestins presented here. This substitution occurs in close proximity to polar core residues - Arg175 in bovine arrestin, which indicates that such changes in electrical charge may alter invertebrate arrestins' structure and binding affinity for unphosphorylated metarhodopsins. Note: squid arrestin (EMBL ID: Q963B5], Drosophila arrestin-1 (EMBL ID: P19107], Limulus arrestin (EMBL ID: P51484], and bovine rod arrestin [EMBL ID: P08168]. The sequences were aligned using NCBI Blast 2 Sequences program.
Squid N'-...367AEPEDYDLIMEEFKRAAVKGFEDEVS"3:':GGLT:'-!6S!q7MGI400-C Limulus N'-...371RMKKQLS:ir'REMS:'81TDLIVEDFARRRQFS396EDNE400-C' Drosophila N' -...361MKKMKS:u;eIEQHRNVKGYYQDDDDNIVFEDFAKMRMNNVNMAD401-C p-arrestinl N' -...381TNDDDIVFEDFARQRLKGMKDDKEEEEDGTGS'J1;!PQLNNR418-C
Figure 9B. Phosphorylation Sites of Arrestins. Phosphorylation sites have been identified in different arrestins [red], and they are conserved at the C-terminal region. The putative sArr phosphorylation sites are S392, T396 andS397. Note: squid arrestin (EMBL ID: Q963B5], Limulus arrestin (EMBL ID: P51484], Drosophila arrestin 2 (EMBL ID: P19107], and B-arrestinl (EMBL ID: P29066]. rod arrestin is also substituted with neutral amino acids in these invertebrate arrestins, although there are neighboring charged residues at some sites. Both substitutions occur in close proximity to a polar core residue, e.g., Arg169 in sArr. Therefore, we expect that these substitutions may have significant structural impact on invertebrate arrestins, which may render them the ability to bind to unphosphorylated metarhodopsin.
Invertebrate rhodopsins can be phosphorylated by GRKs such as SQRK in squid in the light. The functional role of the phosphorylation is unknown, but it may be linked to arrestin dissociation. Studies in Drosophila have illustrated a potential pathway for the inactivation and regeneration of rhodopsin (Alloway, Howard et al. 2000). Light-activated metarhodopsin first recruits arrestin to rapidly desensitize and inhibit phototransduction.
Subsequent binding of receptor kinase phosphorylates metarhodopsin. When metarhodopsin isomerizes back to rhodopsin, arrestin dissociates. It seems that the rhodopsin phosphorylation may play a role in arrestin dissociation, although the isomerization of metarhodopsin may the factor initiating this process. In squid visual system, the rhodopsin phosphorylation adds on negatively charged phosphate groups to the C-terminus of rhodopsin, which will repel the also negatively charged sArr (theoretical pi = 6.1). A mutagenesis study in Drosophila has recently suggested that the phosphorylation of rhodopsin is related to programmed cell death in rdgC mutants
(Kiselev, Socolich et al. 2000). The rdgC gene encodes a Ca2+-dependent phosphatase in
Drosophila (Steele, Washburn et al. 1992). Mutation of this phosphatase may directly inhibit rhodopsin recycling and lead to a built-up of phosphorylated rhodopsin proteins.
Their results also indicate that the accumulation of phosphorylated rhodopsin in the cytosol is the main cause of apoptosis in this cell lineage. However, this finding may represent an extreme case where normal physiological functions in photoreceptors are
26 disrupted.
1.4 Phosphorylation of Arrestin
Phosphorylation of known arrestins has been found to occur at the C-terminal region of these proteins (Figure 9B) (LeVine, Smith et al. 1990; Mayeenuddin and Mitchell
2001). Mammalian p-arrestinl is phosphorylated at Ser412 by the extracellular signal- regulated kinase ERK 1/2, whereas (3-arrestin2 is phosphorylated by casein kinase II at
Thr383 and Ser361 (Lin, Miller et al. 1999; Kim, Barak et al. 2002). In Drosophila, visual arrestin Arr2 is phosphorylated by a Ca2+/Calmodulin-dependent protein kinase at Ser366
(Kahn and Matsumoto 1997). In Limulus, visual arrestin lArr is sequentially phosphorylated at three sites, Ser377, Ser381, and Ser396 (Sineshchekova, Cardasis et al.
2004). Phosphorylation of sArr was consistently observed in our previous studies in a
SQRK-dependent manner (Mayeenuddin and Mitchell 2001; Mayeenuddin and Mitchell
2003; Swardfager and Mitchell 2007). However, the site of phosphorylation on sArr has not been determined. In the current study, we propose that the putative phosphorylation sites are Ser392, Thr396 and Ser397.
Phosphorylation of mammalian p-arrestins does, not affect receptor binding or desensitization assays performed in cells, but the phosphorylation was shown to inhibit arrestin-mediated internalization (Lin, Krueger et al. 1997). p-arrestins are constitutively phosphorylated in the cytosol. Mutation of their phosphorylation sites from serine to alanine did not change arrestin's binding or ability to desensitize B-adrenergic receptors in
HEK293 cells. However, the mutation from serine to aspartic acid, which mimics phosphorylated p-arrestin, did inhibit the recruitment of clathrin and other endocytic factors (Lin, Krueger et al. 1997; Lin, Miller et al. 1999). Since the phosphorylation sites of p-arrestins are close to the C-terminal AP2 and clathrin binding sites, the authors concluded that dephosphorylation of p-arrestins is required for the recruitment endocytic factors and initiation of receptor internalization.
Phosphorylation of invertebrate arrestins has been characterized in Drosophila visual systems by induced mutagenesis in the dArr2 gene. Mutants that were not phosphorylateable were found to form an abnormally stable complex even with inactivated receptors (Alloway, Howard et al. 2000). In contrast, the wild-type dArr2, which was phosphorylated at the activating wavelength, could dissociate from rhodopsin following the isomerization of metarhodopsin at the inactivating wavelength (Alloway,
Howard et al. 2000). Therefore, the phosphorylation of dArr induces its dissociation from inactivated rhodopsin. The effect of phosphorylation on arrestin's binding and dissociating from squid rhodopsin has not been determined but will be investigated in this project.
1.5 Rationale, Research Goals and Hypotheses
Previous work in our laboratory has cloned and purified sArr from squid eyes. We have determined that arrestin is phosphorylated by SQRK. However, several attempts to determine the sites of phosphorylation by mass spectroscopy were unsuccessful. In order to further investigate the role of arrestin phosphorylation, I took the alternative approach in this thesis of expressing recombinant sArr in E. coli and used mutagenesis to test the sites of phosphorylation. The goals of my work were:
1. To express recombinant hexahistidine-tagged wild type sArr in E. coli and
partially purify the recombinant protein using affinity chromatography.
2. To determine if recombinant wild type sArr could bind to visual membranes in
a light-dependent manner. 3. To determine if recombinant wild type sArr could be phosphorylated by
purified SQRK.
4. To determine the site(s) of phosphorylation on sArr using mutagenesis of the
putative sites within the C terminus, S392, T396 and S397.
5. To investigate the effect of phosphorylation on arrestin binding and dissociation
from squid rhodopsins.
My hypotheses that were tested can be stated as follows:
1. sArr is phosphorylated at the C terminus of the protein by SQRK.
2. sArr phosphorylation increases its dissociation from inactive rhodopsins.
2. Materials and Methods
2.1 Materials
Ni-Nitrilotriacetic Acid Agarose resin was purchased from QIAGEN Inc.
Radioisotope [y32-P]ATP (3000 Ci/mmol) was purchased from PerkinElmer Inc. Anti- arrestin polyclonal antibody was previously produced in our lab by inoculating a rabbit with purified sArr (Mayeenuddin and Mitchell 2003). Native squid arrestin (nsArr) and
SQRK were previously purified by Walter Swardfager from Loligo pealei eyes that were purchased from Marine Biology Institute, Woods Hole, MA. Full-length sArr cDNA was cloned in pDONR201 plasmid vector from Invitrogen Inc. (Mayeenuddin and Mitchell
2003).
2.2 Preparation of Dark-Adapted Salt-Washed Rhabdomeric Membranes
All procedures were carried out in the dark under dim red light. Single squid eyes were thawed on ice for 1 hour in 2 mL HEAD buffer (10 mM HEPES pH 7.5,1 mM EGTA, 1 mM AEBSF, 1 mM DTT) containing 500 mM NaCl. The eye structure was dismantled in solution by shaking. Eyecups, lens, and extraneous tissues were removed with forceps, and the remaining solution, containing photoreceptors, was homogenized with a Dounce
Tissue Grinder for 5 strokes. After homogenization, the solution was centrifuged at 10,000 x g for 20 minutes at 4 °C. The pellet, mainly formed by rhabdomeric membranes, was resuspended and dounced twice more in HEAD buffer containing 500 mM NaCl and, lastly in HEAD buffer. The finial pellet was resuspended in 1 mL HEAD buffer and stored at -80
°C.
2.3 cDNA Preparation and Mutation
The full length sArr cDNA, previously cloned, was used as wild type sArr cDNA
(rsArrWT). rsArrWTwas first transferred to vector pDESTl 7 by recombinant LR reactions of the Gateway® Cloning System [Invitrogen Inc). The resulted rsArr-pDESTl 7 plasmid contains an N-terminal hexahistidine tag [2.7 kDa) that enables protein purification by metal affinity chromatography [Appendix). The rsArrWT-pDESTl 7 was also used as a template for generating the following mutants using QuickChange® Site-Directed
Mutagenesis Kit [Stratagene Inc). In general, two types of mutations were created: [1) a triple mutation mutant - rsArr3A [S392A, T396A, and S397A) and [2) three double- mutation mutants - rsArr2Al (S392A and T396A), rsArr2A2 [T396A and S397A), and rsArr2A3 (S392A and S397A). Primers were designed based on Stratagene's online mutagenesis primer designing program10. Primers for rsArr3A-pDESTl 7 (S392A, T396A, and S397A) were S'-cgctggatgaagttgctggaggcctcg ccgcgatgggaat-3', and 5'- attcccatcgcggcgaggcctccagcaacttcatcctcg-3', for sArr2Al-pDESTl 7 [S392A and T396A) were 51'-gttcgaggatgaagttgctggaggcctcgcctcgatggga-3' and S'-tccca tcgaggcgaggcctccagcaacttcatcctcgaac-3', for sArr2A2-pDESTl7 (T396A and S397A) were
10 Mutagenesis primer designing program: http://www.stratagene.com/tradeshows/feature.aspx?rpId=l 18 S'-gaagttagtggaggcctcgccgcgatgggaatataagacc-3' and S'-ggtcttatattcccatcgcggcgaggcc tccactaacttc-3', and for sArr2A3-pDESTl 7 (S392A and S397A) were S'-gttcgaggatgaa gttgctggaggcctcaccgcgatgggaatataagacc-3' and S'-ggtcttatattcccatcgcggtgaggcctc cagcaacttcatcctcgaac-3'. All primers were purchased from Integrated DNA Technologies.
DNA sequencing was used to confirm successful mutations.
2.4 Production of Recombinant Arrestin
Plasmids were transformed into Rosetta(DE3)pLysS cells (Novagen Inc). Ampicillin
(100 ug/ml) was used to select successfully transformed cell colonies. Single colonies were picked to grow in Lysogeny Broth (LB) (Bertani 1951) at 37 °C, 225 rpm overnight. The LB culture was then used to inoculate Terrific Broth (TB) (Tartof and Hobbs 1987) at a ratio of 1:50 (v/v). Cells were then grown either in a shaking incubator at 37 °C and 225 rpm or at 37 °C in the LEX Bubbling System11 developed by the Structural Genomics Consortium of University of Toronto. Expression of sArr was induced by adding 0.4 mM Isopropyl 6-D-
1-thiogalactopyranoside (IPTG), when the optical density of the liquid medium at 600 nm
(OD600) reached 5. After induction, cells were grown at 15 °C overnight. Centrifugation was applied to harvest cells, and collected cell paste was flash frozen in liquid nitrogen and stored at -80 °C.
2.5 Enrichment of Recombinant Arrestin
In the following experiments, all cell and protein samples were kept on ice or at 4
°C. Cell paste was resuspended in binding buffer (50 mM Tris 8.1, 500 mM NaCl, 5 mM B- mercaptoethanol, 2 unit/mL Benzonase, and Protease Inhibitor Cocktail Mix [Sigma,
P8849] at a ratio of 1 mL/20 g cell paste). Cells were lysed using a microfluidizer at
>18,000 psi/stroke. Cell debris was removed by centrifugation at 50,000 *g for 1 hour.
11 Introduction of the LEX Bubbling System: http://sgc.utoronto.ca/SGC-WebPages/Technology/lex.php The supernatant was mixed with Ni-NTA agarose beads (50 % slurry in binding buffer) at a ratio of 200:1 (v/v). After 2-hour incubation, the Ni-NTA beads were isolated by centrifugation at 500 xg for 3 minutes and washed twice in binding buffer. Bound proteins were eluted from the beads using elution buffer (50 mM Tris 8.1, 500 mM NaCl, 500 mM
Imidazole), and dialyzed overnight in dialysis cassettes (Thermo Scientific's Pierce) in dialysis buffer (50 mM Tris 8.1, 300 mM NaCl, 0.5 mM DTT) (1:400 v/v). The final enriched protein solution was concentrated to >3 mg/mL of total protein. Arrestin expression was confirmed by Gel Electrophoresis and Western blotting with anti-sArr antibody. Total protein yield was measured by Amido Black staining (Schaffner and Weissmann 1973).
Arrestin yield was estimated by comparing its band optical density to that of purified nsArr on Western blots.
2.6 Arrestin Phosphorylation Assays
These assays were carried out as previously described with minor modification
(Mayeenuddin and Mitchell 2001; Swardfager and Mitchell 2007). Enriched sArr preparations (containing 1 ug sArr) were incubated with SQRK (5 ug) for 15 min at 25 °C in phosphorylation buffer (50 mM Tris, pH 8.0,15 mM MgS04,1 mM EGTA, 1 mM ATP, 2 mM DTT, 100 uM ouabain, and 1 uM GTPyS) with [y32P]ATP (> 200,000 cpm/uL), in a total reaction volume of 30 uL. The reaction was terminated by the addition of 30 uL 2x SDS-
PAGE sample buffer. Samples were resolved on 11% SDS-polyacrylamide gels, stained with Coomassie blue dye, de-stained and extensively washed in H2O prior to drying and exposing the X-ray films in autoradiography.
2.7 Membrane Association Assays
The association assay was carried out as previously described with minor modification (Swardfager and Mitchell 2007). Enriched arrestin (3 ug) was incubated with dark-adapted salt-washed membranes (30 ug] in light or dark condition for 15 min at 25°C in association buffer (50 raM Tris, pH 8.0,1 mM EGTA, and 0.5 raM inositol hexakisphosphate) in a total volume of 60 uL. The mixture was centrifuged at 10,000 xg for 15 min at 25°C in the dark. Supernatants were diluted with 60 uL 2x SDS-PAGE sample buffer and pellets were resuspended in 120 uL lx SDS-PAGE buffer. All samples were then resolved on 11% SDS-polyacrylamide gels, and proteins were detected by either autoradiography as described in section 2.6, or anti-sArr antibodies in Western blotting.
2.8 Membrane Dissociation Assays
Enriched arrestin (3 ug] was phosphorylated by SQRK in the light as described in section 2.6. Phosphorylation was stopped by the addition of 20 mM EDTA. The solution was incubated with 30 ug of dark-adapted, salt-washed membranes in the light for 15 min at 25 °C. Different concentrations of NaCl were then added to the mixture, and the total volume of each sample was adjusted to 60 uL with H2O. All samples were dark adapted for
1 hour at 25 °C and then, subjected to centrifugation at 10,000 xg for 15 min at 25°C in the dark. Supernatants were diluted with 60 uL 2x SDS-PAGE sample buffer and pellets were resuspended in 120 uL lx SDS-PAGE buffer. All samples were then resolved on 11% SDS- polyacrylamide gels (at 60 uL/well), and arrestin proteins were detected by Western
Blotting with anti-sArr antibodies.
2.9 Other Methods
Electrophoresis of protein samples was carried out by the method of Laemmli
(Laemmli 1970] on 11 % polyacrylamide gels. In Western blotting, proteins were transferred to nitrocellulose for identification and developed with horseradish peroxidase-conjugated secondary antibody and visualized using Enhanced
Chemiluminescence Substrate (GE Healthcare Bio-Sciences). Western blots were scanned using a Cannon scanner (N670U), and the optical density of bands was determined using
ImageQuant® software. Results were analyzed by Prism software (GraphPad Software
Inc., San Diego, CA, USA]. Significant differences between sample values were assessed by
T-Tests or a liner regression analysis with 2-way ANOVA.
3. Results
3.1 Solubility of Recombinant sArr
Previous attempts to produce recombinant wild type sArr (rsArrWT) in E. coli cells had shown low solubility of the protein in aqueous buffers (Mayeenuddin and Mitchell
2003; Swardfager and Mitchell 2007). However, a systematic approach to test rsArrWT solubility under multiple conditions was not undertaken. One of the key factors that may result in low recombinant protein solubility is the rate at which the protein is being produced in bacterial cells. Simply put, when the recombinant protein is produced at a higher rate than can be processed by the protein folding mechanism, large amounts of the overexpressed proteins would become misfolded and aggregate. The production rate may be also the key factor determining recombinant arrestin's solubility. Therefore, my initial goal was to optimize the rsArrWT production rate so to improve its solubility. I tested various conditions including IPTG induction concentration, cell growth temperature, bacterial cell line, affinity tag, and pH during chromatography. These results are described below.
Before utilizing Rosetta (DE3)pLysS cells, I tested sArr solubility with BL21(DE3)-
SI cells which was a type BL21(DE3) cells induced by NaCl. The soluble portion of sArr was very low, potentially due to protein aggregation (results not shown). The extensive presence of NaCl in growth media may induce the expression of T7 RNA polymerase, initiating target protein expression prior to induction. The proteins produced tend to aggregate and precipitate, given that the majority of them are formed at 37 °C - a suboptimal condition that may exacerbate protein denaturation. On the other hand,
Rosetta (DE3)pLysS cells have incorporated T7-specific lysozyme genes in their genome, so that uninduced T7 RNA polymerase and subsequent protein expression can be effectively inhibited. However, the usage Rosetta (DE3)pLysS cells did not significantly increase sArr solubility, as illustrated in the following sections.
In the Rosetta (DE3)pLysS cells we used, IPTG induces the production of T7 RNA polymerase which in turn induces the recombinant sArr production. By varying the IPTG induction concentration, the production of recombinant sArr may be changed. In Figure
10A, we showed that within the common range of IPTG induction concentrations (0.2-1.2 mM], the majority of rsArrWT precipitated and was found in the insoluble pellets (PEL).
The soluble portion of rsArrWT (as shown in the ELU lanes) was extremely low. Lower concentrations of IPTG were not tested because within the range of 0.2-1.2 mM, I did not observe any correlation between IPTG concentration and rsArrWT solubility. However, there was a lower total amount of rsArrWT in samples induced with 0.2 mM compared to
1.2 mM IPTG. Therefore, I used a concentration of 0.4 mM IPTG in all subsequent experiments, unless specified, as a compromise between solubility and arrestin yield.
Cell growth temperature is another way to optimize the protein production rate.
The lower the temperature, the slower the recombinant protein is produced. As shown in
Figure 10B, we tested growth temperatures of 15 °C, 20 °C and 30 °C, and achieved similar results in all three cases, again with the majority of rsArrWT precipitated. At this stage, it seemed simply controlling the protein production rate was not an effective way of increasing rsArrWT solubility. 0 0.2 0.4 0.8 1.2 0.2 0.4 0.8 1.2 IPTG(mM)
55kD <-rsArrWT
-ELU-
B 15 20 30 15 20 30 15 20 30 15 20 30 Temp (°C)
*w»
H$l£ !"(•'• 4&&V& - -rU&ttfe III iMflmlllflfri rg&Hl
55kD orsArrWT
[—-PEL-—] [-—SUP-—] [ --WA --] [—ELU--]
Figure 10. Solubility of rsArrWT at Different IPTG Induction Concentrations and Growth Temperatures. rsArrWT-pDESTl 7-transformed Rosetta(DE3)pLysS cells were grown in LB at 37 °C to reach an OD600 of 0.5. Cells were induced with either IPTG at 4 different concentrations at 20 °C (A), or 0.4 mM IPTG at different temperatures (B), and harvested when their OD600 reached 1. Harvested cells were lysed in binding buffer (yielding whole cell lysate - WCL) and then centrifuged at 10,000 xg to remove insoluble pellets (PEL). Supernatants (SUP) were extracted with Ni2+-beads for His-tagged rsArrWT. The beads were washed in binding buffer (WA), and Ni2+-bound proteins were eluted in the elution buffer (ELU). All samples were resolved by SDS-PAGE (4-20 % polyacrylamide gradient gel) and stained with Simple Blue™ (A) or Coomassie blue (B). Resuspended PEL was loaded at 5 ug/lane and other samples at 10 uL/lane. rsArrWT ran at approximately 55 kDa as indicated. I then tested rsArrWT solubility in a modified BL21cell line - Origami B cells. The
Origami B cell hosts additional plasmids that carry mutations in both the thioredoxin
reductase and glutathione reductase genes, which are known to enhance disulfide bond
formation between cysteines [Bessette, Aslund et al. 1999]. The disulfide bond may also
contribute to protein folding and solubility. Since there are 8 cysteines in rsArrWT, there is
a significant chance that some of these cysteines would form disulphide bonds. In Figure
11,1 demonstrated that rsArrWT solubility was not improved in Origami cells, and again
the majority of the rsArrWT precipitated.
I next tried adding protein tags- Glutathione S-Transferase (GST) and Maltose-
binding protein (MBP) tags to the rsArrWT. The GST-tag was added at the N-terminus of
sArr as in the recombinant vector sArrWT-pDEST15. The MBP tag was also added at the N-
terminus of sArr as in recombinant vector sArrWT-pDESTHisMBP (Appendix). The
construction of all recombinant vectors used is illustrated in the appendix. Both GST and
MBP tags have been reported to improve protein solubility by retaining target proteins in
the cytosol where the tags are soluble. However, in the case of rsArrWT, neither the GST
nor MBP tag significantly improved rsArrWT solubility, as shown in Figure 12.
The last condition I tested was the pH of the binding buffer for arresting binding to
Ni2+-NTA beads. A practical rule to choose the buffer pH for protein purification is the
theoretical pi of the target protein +/-1. The theoretical pi of rsArrWT was 7.1 including
the His-tag. Therefore, I set my binding buffer at pH 8.1. The reason I did not choose 6.1 or
below was to avoid the negative effects of acidic conditions on Ni2+-protein binding. I also tested pH 8.5 for the binding buffer. As shown in Figure 13, there was no difference in the
amount of soluble rsArrWT at the two pH values. A 0 0.2 0.4 0.8 1.2 0 0.2 0.4 0.8 1.2 IPTG (mM)
55kD I <-rsArrWT
-WCL- ] [" -SUP-
B 0 0.2 0.4 0.8 1.2 0 0.2 0.4 0.8 1.2 IPTG (mM)
SSkD <-rsArrWT
-PEL- ] [- -ELU-
Figure 11. Solubility of rsArrWT in Origami Cells at Different IPTG Induction Concentrations. rsArrWT-pDESTl 7-transformed Origami™ B Cells were grown at 37 °C to reach an OD600 of 0.5. Cells were then induced with IPTG at 4 different concentrations at 20 °C, and harvested when the OD600 reached 1. Samples were prepared as described in Figure 10. All samples were subjected to SDS-PAGE [4-20 % polyacrylamide gradient gel) and stained with Simple Blue™. Both WCL and PEL were loaded at 5 ug/lane and other samples at 10 uL/lane. rsArrWT ran approximately at 55 kDa as indicated. 0 0.2 0.4 0.8 1.2 0.2 0.4 0.8 1.2 IPTG(mM)
70kD •mm ***** SS? 152! 125 IK? «•» <-GST-rsArrWT Mi,
lifewwjSrfSfe, «pw»
-PEL- -ELU-
B 0 0.2 0.4 0.2 0.4 0.2 0.4 0.2 0.4 IPTG(mM)
t»&j«Sk 9M«*S lOOkD f0§fc W*«* <-MBP-rsArrWT
-WCL- ] [ -SUP -] [--PEL—] [ -ELT---]
Figure 12. Solubility of GST- and HisMBP-Tagged rsArrWT at Different IPTG Induction Concentrations. Plasmids rsArrWT-pDESTIS and rsArrWT-HisMBP were transformed to Rosetta(DE3)pLysS cells and grown at 37 °C to reach an OD600 of 0.5. Cells were induced with IPTG at indicated concentrations at 20 °C and harvested when the OD600 reached 1. All samples were subjected to SDS-PAGE (4-20 % polyacrylamide gradient gel) and stained with Simple Blue™. For GST-tagged samples, the PEL was loaded at 5 ug/lane and the ELU at 10 uL/lane (A). All HisMBP-tagged samples were loaded at 10 uL/lane (B). GST-rsArrWT (75.3 kDa) and HisMBP-rsArrWT (87.3 kDa) ran at approximately 70 kDa and 100 kDa as indicated. 8.1 8.5 8.1 8.5 8.1 8.5 8.1 8.5 8.1 8.5 pH
r -\ ps ri Hium !*?**2p&' 5E 'S^-S,
55kD mm jf"1**| ft <-rsArrWT
ym^*t w^m
-WCL- -] [--SUP-] [-PEL-] [-ELU-]
Figure 13. Effects of Lysis Buffer pH on rsArrWT Solubility. rsArrWT-pDEST17- transformed Rosetta(DE3)pLysS cells were grown at 37 °C to reach an OD600 of 0.5. Cells were then induced with 0.4 mM IPTG (except for the first 2 lanes which were uninduced controls) at 20 °C and harvested when their OD600 reached 1. Harvested cells were lysed and extracted by Ni2+-beads in the binding buffer with pH values of 8.1 or 8.5. All samples (at 10 uL/lane) were subjected to SDS-PAGE (4-20 % polyacrylamide gradient gel] and stained with Simple Blue™. rsArrWT ran at approximately 55 kDa. Previous work in our lab had extracted recombinant arrestin in urea and tried various protocols to refold the protein. Although this process did yield pure rsArrWT, these proteins were not functional in our phosphorylation assays (unpublished results).
Therefore, the denaturation and refolding of arrestin was not repeated here.
3.2 Recombinant sArr Production and Enrichment
The final approach that I took was to maximize the amount of soluble protein was to scale up production of recombinant sArr in at least 6 L volumes. rsArrWT, rsArr3A
(S392A, T396A and S397A) and rsArr2A3 (S392A and S397A) were enriched as illustrated in Figures 14,15, and 16, respectively. As shown in these figures, small amounts of enriched recombinant arrestins were obtained after affinity chromatography with Ni2+ beads; however, protein yields were too low to attempt additional steps of purification. I could not obtain enough soluble proteins for the other two mutants, rsArr2Al and rsArr2A2 for my experiments. A possible explanation for the very low yield of these two arrestin mutants was the change in cell lines required at the Structural Genomics
Consortium due to phage contamination of the facility. Although the new phage-resistant cell line was derived from the same BL21(DE3] cells, the cells seemed to yield much less soluble arrestins even on a larger growth scale. Further attempts to produce these two mutants were therefore abandoned.
The identity of rsArr was confirmed by Western blotting using anti-sArr antibodies and the rsArr could also be identified on Coomassie or Simple Blue stained polyacrylamide gels at 55 kD region, consistent with the native sArr (nsArr) purified in our lab
(Swardfager and Mitchell 2007). The purity of arrestin was estimated by comparing its band density to the lane density in Coomassie or Simple Blue stained gels. The estimation was also confirmed by comparing the optical density of Western blot bands of rsArr and A B
Figure 14. Representation of the rsArrWT Enrichment Process. Details of the enrichment process were described in the Method section. All samples were subjected to SDS-PAGE (4-20 % polyacrylamide gradient gel) and stained with Simple Blue™ (A) or subjected to Western blotting with anti-sArr antibodies (B & C). Each lane was loaded with 5 ug proteins. Total protein concentrations in final elution was 5.6 ug/uL, of which the rsArrWT accounted for 15+4 %, estimated by comparing band density of rsArrWT to that of the total protein. The estimation was confirmed in Western blot when rsArrWT and nsArr were loaded at 1 ug/lane (B). Abbreviations: WCL - Whole Cell Lysate, PEL - Pellet, SUP - Supernatant, FT - Flow Through, and ELU - Elution. 55kD Sjg^gg^ .DM
WCL PEL SUP ELU
B
55kD
WCL PEL SUP FT ELU
Figure 15. Representation of the rsArr3A Enrichment Process. All samples were subjected to SDS-PAGE (4-20 % polyacrylamide gradient gel) and stained with Simple Blue™ (A) or subjected to Western blotting with Anti-sArr antibodies (BJ. Each lane was loaded with 5 ug proteins. Total protein concentrations of the final elution was 3.5 ug/uL, of which the rsArr3A accounted for 11±3 %, estimated by comparing band density of rsArr3A to that of the total protein. ttfif/IHlt
55kD tt*m*t <-rsArr2A3
|MWf
WCL PEL SUP FT ELU
B
rsArrWT rsArr3A rsArr2A3
Figure 16. Representation of the rsArr2A3 Enrichment Process. All samples were subjected to SDS-PAGE (4-20 % polyacrylamide gradient gel, at 5 ug/lane) and stained with Simple Blue™ (A]. The samples of rsArrWT, rsArr3A and rsArr2A3 were subjected to Western blotting with Anti-sArr antibodies, and each lane was loaded with 3 uL of protein samples. Protein concentrations of rsArr2A3 was 6.9 ug/uL, of which the rsArr2A3 accounted for ~9 %, estimated by comparing band density of rsArr2A3 to that of the total protein. nsArr at equivalent protein concentrations. As listed in Table 1, the average purity of
rsArrWT, rsArr3A and rsArr2A3 were 15±4 %, 11±3 % and 9 % of total proteins,
respectively.
Table 1. Estimation of arrestin concentration and yield per 120 g cell paste.
Total Protein Arrestin to Total Average Arrestin Concentration Protein Ratio Concentration Average Yield
rsArrWT 5.6±0.5 ug/uL 15±4 % 0.8 ug/uL 2.5 mg
rsArr3A 3.5±0.3 ug/uL 11±3 % 0.4 ug/uL 1.2 mg
rsArr2A 6.9 ug/uL 9% 0.6 ug/uL 1.9 mg
3.3 Phosphorylation of Recombinant sArr
3.3.1 SQRK-Dependent sArr Phosphorylation
Enriched rsArrWT was phosphorylated by reconstitution with purified SQRK as shown in Figure 17. In comparison to the phosphorylation of purified nsArr, rsArrWT phosphorylation was significantly lower as measured by the band density in the autoradiograph of Figure 17A. This suggests that a portion of the enriched rsArrWT was not properly folded or recognized by SQRK. As the total arrestins in nsArr and rsArrWT are the same as shown by the Western blot (Figure 17B), approximately 26 % of rsArrWT may not be phosphorylated by SQRK. Nonetheless, the majority of the enriched rsArrWT was phosphorylated in a SQRK-dependent manner.
It should be noted that the light phosphorylation band observed in the SQRK lane in
Figure 17A is a small amount of sArr contaminant in the SQRK preparation, as confirmed by the Western blot (Figure 17B). The other light phosphorylation band found with nsArr alone is the result of a small amount of radioactive phosphates bound to nsArr as previously suggested (Swardfager and Mitchell 2007]. These residual phosphorylations 55kD
SQRK nsArr rsArrWT nsArr+ rsArrWT SQRK +SQRK
900 * a 750 o •M 600 t/1 I—* e 2 °s 450 u •— M S 300 .2 = « s- 150 o 3 0 nsArr+SQRK rsArrWT+SQRK
B
55kD
SQRK nsArr rsArrWT
Figure 17. SQRK-Dependent sArrWT Phosphorylation. In panel A, both nsArr and rsArrWT were phosphorylated by SQRK, but the nsArr phosphorylation was significantly higher than the rsArrWT phosphorylation (* n=3, p<0.05, T-Test). The autoradiograph also showed background phosphorylation in SQRK and nsArr lanes. The SQRK background was subtracted from both nsArr and rsArrWT phosphorylations, and the nsArr background from the nsArr phosphorylation. In panel B, Western blot with anti-sArr antibodies conformed equal loading of nsArr and rsArrWT, and the presence of nsArr in SQRK preparations. were subtracted from the sample phosphorylations. All recombinant proteins had a 2.7 kD hexahistidine-tag making them run slightly higher than the nsArr on Western blots [Figure
17B).
3.3.2 Membrane and Calcium Effects on sArr Phosphorylation
The presence of membranes significantly increased the SQRK-dependent rsArrWT phosphorylation, Figure 18. This finding confirmed our previous results that the presence of membrane significantly increased nsArr phosphorylation (Swardfager and Mitchell
2007). As shown in the same figure, the phosphorylation of rhodopsin was also SQRK- dependent. The phosphorylation of sArr was also observed in the absence of membrane preparations, which was absent in previous studies. The implication of this discrepancy will be discussed in the discussion section.
Previous experiments with native sArr either on the membranes or reconstituted with membranes following purification found that calcium increased sArr phosphorylation. However, the presence of Ca2+ in our assays did not change rsArrWT phosphorylation when reconstituted with SQRK alone, Figure 19. The calcium concentration in squid photoreceptors has been reported to range from 0.5 uM in the inactive state to 100 uM in the light-activated state (Rack, Xhonneux-Cremers et al. 1994).
Both previous and current studies have used 100 uM calcium to test its potential effects on sArr phosphorylation. This was the concentration of calcium that induced a maximal increase in phosphorylation in our previous dose-response studies [Mayeenuddin and
Mitchell 2003).
3.4 Determination of the Sites of Phosphorylation on sArr
Based on studies on other arrestins, the most probable sites of phosphorylation on sArr are in the C-terminus at Ser392 and Ser397 or Thr396. To test this hypothesis, I first 55kDi
43kDl
180 u * 0> 4-1 150 0) E 4-o1 •FN 120 GC/J /—2> Q © 90 u. *—< -* TJ2 W) C 60 •£=a > « I* o 30 s
rsArrWT+SQRK rsArrWT+SQRK+MEM
Figure 18. Membrane Effects on rsArrWT Phosphorylation. In the presence of membranes (MEM), rsArrWT phosphorylation was significantly increased, as shown in the upper autoradiograph lower bar graph as mean ± SEM (* n=3, p<0.05, T-Test). 55kD
SQRK rsArrWT nsArr+ rsArrWT+ rsArrWT+ SQRK+Ca2+ SQRK-Ca2+ SQRK+Ca2+
3UU
mete r 400 -
insito i DO ) 300 - ^m WJM aphD i t (xlO l 200 - ^m ^M Un i
adiog r 100 - ^m ^M
Auto r o - ^^^^^^w t,,, rsArrWT+SQRK rsArrWT+SQRK -Calcium +Calcium
Figure 19. Calcium Effect on rsArrWT Phosphorylation. The phosphorylation of rsArrWT was represented in the upper autoradiograph and the lower graph represents band density mean ± SEM. The CaCl2 was added at 100 uM and no significant difference was found between with and without CaCl2 (n=3, p>0.05). SQRK lane indicates phosphorylation of potential nsArr contaminant; rsArrWT land indicates phosphorylation in the absence of SQRK; nsArr+SQRK+Ca2+is the positive control for SQRK activity. made a recombinant sArr mutant (rsArr3A] with all three amino acids Ser392, The396, and
Ser397 mutated to alanines. In reconstitution with SQRK, rsArr3A showed 94 % reduction in phosphorylation in comparison to the rsArrWT phosphorylation, Figure 20A. The residual phosphorylation of rsArr3A was indistinguishable from the phosphorylation in the absence of SQRK [Figure 20A). Therefore, the mutations completely inhibited SQRK phosphorylation of sArr. I next tested phosphorylation of rsArr2A3, with alanine mutations at Ser392 and Ser397, in the same reconstitution assay with SQRK, Figure 21A. It again demonstrated complete loss of SQRK-dependent phosphorylation. The residual phosphorylation of rsArr2A3 by SQRK did not show significant difference from that of rsArr3A. Therefore, we conclude that the phosphorylation sites of squid arrestin are Ser392 and/or Ser397.
3.5 Membrane Binding Assays
The effect of arrestin phosphorylation on the protein's function can be divided into two aspects; one, the effect on arrestin's association with metarhodopsin and, two, the effect on arrestin's dissociation from inactivated rhodopsin. We first demonstrated the arrestin's binding to metarhodopsin using membrane preparations. In our reconstitution assays, both rsArrWT and rsArr3A bound membranes in a light dependent manner illustrated in Figure 22. However, the binding to metarhodopsin (in the light condition) of rsArr3A was significantly higher than that of rsArrWT, suggesting that the serines and threonine may contribute to arrestin's binding affinity to metarhodopsin.
Because of the low membrane binding of rsArrWT, I opted to use purified nsArr in the following assays. To determine any potential phosphorylation effects on sArr binding to membranes, I first phosphorylated nsArr by SQRK. Phosphorylations were performed with radioactive ATP to trace only the phosphorylated nsArr by autoradiography. The 55kD
nsArr rsArr3A rsArrWT SQRK rsArr3A rsArrWT +SQRK +SQRK +SQRK
500
rsArr3A rsArrWT rsArr3A+SQRK rsArrWT+SQRK
B
55kD
rsArr3A rsArrWT
Figure 20. Phosphorylation of rsArrWT and rsArr3A. As shown in panel A, the phosphorylation of rsArrWT was quantified by ImageQuant. The results were represented as meantSEM (* n=3, p<0.01, T-Test}. In panel B, the Western blot with anti-sArr antibodies confirmed that both rsArrWT and rsArr3A were equally loaded in the assay. 55kD?
'.,: »j^^^™»^»J-^*^*^^iSp^*^>j-\-**:s -v
SQRK rsArr2A3 rsArrWT rsArr3A rsArr2A3 +SQRK +SQRK +SQRK 1200
rsArr2A3 rsArr3A+SQRK rsArr2A3+SQRK rsArrWT+SQRK
B
55kD
nsArr rsArrWT rsArr3A rsArr2A3
Figure 21. Phosphorylation of rsArrWT, rsArr3A and rsArr2A3. As shown in panel A, the phosphorylation band density on the autoradiograph was quantified by ImageQuant software. The results were represented as mean±SEM (* n=3, p<0.01, T-Test). In panel B, the Western blot with anti-sArr antibodies confirmed all samples were equally loaded. A B
55kD
SUP(L) PEL(L) SUP(D) PEL (D) SUP(L) PEL(L) SUP(D) PEL (D)
80 •o a 3 M # a> _- 60
55 aflj 40 o St O r^ * r 20 « s •5 "> c S rsArrWT+MEM rsArrWT+MEM rsArr3A+MEM rsArr3A+MEM (L) CD] CL) (D)
Figure 22. Light-Dependent Membrane Binding of rsArrWT and rsArr3A. Dark-adapted membranes (MEM) were incubated with rsArrWT (panel A) or rsArr3A (panel B), and the mixtures were subjected to either light (L) or dark (D) for 15 minutes and then centrifuged to yield supernatants (SUP) and pellets (PEL). All samples were resolved by SDS-PAGE and transferred to nitrocellulose for detection with anti-sArr antibodies in Western blotting. Binding ratio of the membrane-bound to total arrestins was calculated by the following formula: „ . ^ --„,,, Densitometers of PEL % Densitometers of (PEL + SUP)
The result was represented as mean±SEM, (* n=3, p<0.05 and ** n=3, p<0.01, T-Test). phosphorylation was terminated by addition of EGTA, and samples were then bound to dark-adapted membranes in either the dark or the light. The ratio of membrane bound to total nsArr is illustrated in Figure 23A. The phosphorylated nsArr bound to membranes in a light-dependent manner, with a 3.4 fold increase from dark to light, comparable to that of the unphosphorylated rsArrWT but lower than that of rsArr3A. As shown in Figure 23A, higher background binding of nsArr (26±2 %) than rsArr3A (13±4 %) may be the main reason for the smaller fold increase for nsArr. The high background binding is likely caused by sedimentation after long-term storage and/or arrestin binding to lipids as previously suggested [Swardfager and Mitchell 2007).
The binding of total nsArr to membranes, phosphorylated and unphosphorylated, was assayed in the same way as described above, but instead of using autoradiography,
Western blotting with anti-sArr antibodies was used to detect all nsArr. As shown in
Figure 23B, the total nsArr regardless of phosphorylation status binds to membranes in the light with comparable ratios, 80±6 % and 89±4 % for the phosphorylated and unphosphorylated nsArr, respectively.
In Figure 24, a comparison of membrane binding is made between the radiolabeled phosphorylated nsArr and unphosphorylated total nsArr. This comparison confirms that around 90 % nsArr bind to membranes in the light regardless of phosphorylation.
However, when nsArr is phosphorylated, the background (dark) binding is significantly reduced, suggesting that the phosphorylation may inhibit membrane binding prior to light-activation.
3.6 Membrane Dissociation Assays
Increasing the salt concentration in the dissociation buffer was shown in our previous studies to facilitate nsArr dissociation from dark-adapted membranes in the SUP(L) PEL(L) SUP(D) PEL (D)
IOO fc ^^^^ ** £ 75 oSag C t 4J H 50 *•MO .a Wa S ft «) | 25 CL- Jfcztt: "p-nsArr+MEM (L) *p-nsArr+MEM (D)
Ess&issy
SUP(L) PEL(L) SUP(D) PEL(D) SUP (L) PEL(L) SUP(D) PEL(D) -ATP -ATP -ATP -ATP +ATP +ATP +ATP +ATP
s- uu H ^^^^ ** u | ** < 7c; - ^^^^| / O 1 a ^^M o o K(\ - ^H O O) 0i "o DID. TK - H g sw •3 c 0 - ^^^^^" i ^^^^^P inmiMim 5 nsArr+MEM (L) nsArr+MEM (D) p-nsArr+MEM (L) p-nsArr+MEM (D)
Figure 23. Effects of Phosphorylation on nsArr Binding to Membranes. In panel A, phosphorylated nsArr was labeled with [y32-P]ATP and detected by autoradiography. The membrane-bound to total nsArr ratio was presented as mean ± SEM (** n=3, p<0.01, T-Test). In panel B, total nsArr binding was detected by Western blotting with anti-sArr antibodies, and the membrane bound to total nsArr ratio was presented as mean + SEM (** n=3, p<0.01, T-Test). *p-nsArr: radiolabeled phosphorylated nsArr, p-nsArr: phosphorylated nsArr.
55 100
nsArr+MEM *p-nsArr+MEM nsArr+MEM *p-nsArr+MEM (L) (L) (D) CD)
Figure 24. Comparison between phosphorylated and unphosphorylated nsArr. This figure was adapted from Figure 23A and B, with comparison between the phosphorylated and unphosphorylated nsArr in either light or dark condition. The binding ration of membrane bound to total nsArr were represented by mean ± SEM (* n=3, p<0.05, T-Test) process of protein purification (Swardfager and Mitchell 2007]. In this assay, we further explored the effect of phosphorylation on nsArr dissociation from membranes in the dark.
Samples of nsArr were incubated with SQRK in the presence or absence of ATP to create phosphorylated or unphosphorylated nsArr. The proteins were then incubated with membranes in the light for 5 minutes to bind arrestins to metarhodopsin and then moved to the dark and supplemented with increasing amounts of NaCl. After one hour in the dark, membranes were centrifuged to separate the membrane-bound and unbound arrestins. All samples were then resolved by gel electrophoresis and transferred to nitrocellulose membranes for Western blotting with anti-sArr antibodies. The binding ratio of membrane-bound to total nsArr was plotted against the NaCl concentration, as shown in
Figure 25. In this assay, we demonstrated that nsArr dissociation from dark-adapted membranes was increased with increasing NaCl concentrations. The dissociation curves of phosphorylated and unphosphorylated nsArr are fitted to a non-liner regression model using the GraphPad Prism software. When nsArr was phosphorylated, the dissociation curve significantly shifted to the left. The NaCl concentrations for 50 % dissociation were
584 mM and 883 mM for the phosphorylated and the unphosphorylated nsArr, respectively. These results suggest that the phosphorylation of sArr may increase the protein's dissociation from inactivated rhodopsin or dark-adapted membranes. • UnphosphoiylatediisArr • Phosphorylated nsArr
2.00 2.25 2.50 2.77 2.95 Log [NaCI] mM
Figure 25. Dissociation of nsArr from Dark Adapted Membranes. nsArr proteins were first incubated with SQRK in phosphorylation buffers with or without 2mM ATP for 15 minutes. The samples were then bound to membranes in light for 5 minutes. Then the mixtures were dark adapted for 1 hour at room temperature with various NaCI concentrations. All samples were subjected to centrifugation to separate membrane bound and unbound nsArr. nsArr were detected by Western blotting with anti-sArr antibodies. The membrane bound to total nsArr ratio was plotted against LOG NaCI concentrations, as shown above. The dotted line indicated where 50 % of nsArr dissociated from membranes (DC50). The NaCI concentrations at the DC50 were 584 mM and 883 mM for phosphorylated and unphosphorylated nsArr, respectively. These values as well as the two dissociation curves were significantly different in the non-liner regression and inhibition model, with p<0.05. 4. Discussion
Our previous studies have successfully cloned and purified sArr from Loligo pealei eyes and characterized its major function in the squid visual system. The nsArr binds to membranes in a light-dependent manner and inhibits the interaction between metarhodopsin and iGqoc, resulting in a significant loss of the GTPase activity of iGqa. The nsArr was identified initially as a SQRK substrate (Mayeenuddin and Mitchell 2001), and later shown to be phosphorylated by SQRK in reconstitution assays with membranes
(Swardfager and Mitchell 2007). However, the sites of phosphorylation and its functional role have not been determined.
My study has used recombinant sArr, mutagenesis methods and purified nsArr to determine the sites and functional role of arrestin phosphorylation in interacting with rhodopsin. The following 3 major findings were presented in this thesis.
1. By growing the s/lrr-transformed E. coli cells on a large scale, I was able to
partially purify recombinant sArr proteins for functional analyses.
2. The enriched rsArrWT was phosphorylated by purified SQRK alone in
reconstitution assays. In addition, the SQRK-dependent phosphorylation was
completely inhibited when Ser392 and Ser397 were mutated to alanines,
indicating that the Ser392 and/or Ser397 were the phospohrylation sites on sArr.
3. Both recombinant and native sArr bound to membranes in a light-dependent
manner. Phosphorylation of nsArr did not affect the protein's association with
membranes in the light, but significantly increased its dissociation from dark-
adapted membranes.
4.1 Recombinant sArr Production and Purification Producing recombinant proteins in E. coli cells is an efficient and economical way to obtain a large amount of pure protein. However, the bacterial expression system is not suitable for all proteins, especially eukaryotic proteins either encoded by specific tRNA codons or requiring post-translational modifications. Various factors are responsible for expressing, folding and processing to yield the final protein products. In the current study, in order to increase recombinant sArr solubility I tested multiple conditions including
IPTG induction concentration, growth temperature, addition of a protein tag, different bacterial cell lines, and binding buffer pH values. However, regardless of the condition of cell growth and the route of protein purification, the majority of rsArr precipitated in aqueous solution. It is unclear which conditions would induce or reverse the precipitation process. Given that the native sArr is a soluble protein in the photoreceptor, it is most likely that the insoluble recombinant sArr is not correctly folded in E. coli, resulting in aggregation and formation of inclusion bodies inside the bacteria. The formation of inclusion bodies has been one of the common difficulties in obtaining recombinant proteins from bacterial cells. The inclusion bodies are composed of densely packed denatured proteins, and often their components are highly homogenous - formed mostly by overexpressed target proteins (Singh and Panda 2005). The mechanism by which the inclusion body is formed is not fully understood, but it may depend on the competition between target protein folding and aggregation (Fahnert, Lilie et al. 2004). The rate of protein folding is determined and limited by structural characteristics of individual proteins. If a protein has a distinct structural feature that can not be correctly folded by E. coli, the protein is more susceptible to forming inclusion bodies and remains insoluble regardless of growth and purification conditions. This appears to be the case for recombinant sArr. The aggregation rate is proportionally linked to the protein production rate by host cells. In most E. coli cell-based protein production systems, such as the one I used here - driven by T7-polymerases, the target protein production rate is high.
Therefore, high expression rate may further exacerbate recombinant sArr misfolding. In many cases, controlling the production rate has been effective in increasing protein solubility. However, in my studies, the various conditions used to slow down protein production were not helpful. These observations suggest that recombinant sArr may require specific folding mechanisms or post-translational modifications, which are not available in E. coli cells.
One of the methods that may significantly enhance the folding capabilities of E. coli cells is the co-production of heat-shock chaperone protein with recombinant proteins. The heat-shock chaperones are a group of proteins ubiquitously expressed in all types of cells
(Marston 1986). Their production is highly inducible by stressful conditions such as elevated temperatures, and they are known to help protein folding and prevent protein aggregation (Marston 1986). Although the chaperons do exist in E. coli cells, they may be overwhelmed by production of exogenous proteins under the control of a strong promoter at temperatures < 37 °C. Co-transformation of bacterial chaperone genes has been reported as an effective way to improve the solubility of some proteins (Hoffmann and
Rinas 2004). Future tests of chaperone protein co-expression with recombinant sArr may yield desirable results.
The other protein expression system that should be considered is the baculovirus- insect cell expression system. The insect cell-based system can produce, fold and modify eukaryotic proteins in a way that is close to their native conditions (Kost, Condreay et al.
2005). However, it should be noted that post-translational modification by phosphorylation has been reported in the insect cell (Hoss, Moarefi et al. 1990). The uncontrolled phosphorylation of sArr would obviously be a problem when we are testing the effect of SQRK phosphorylation and therefore tests for the presence of phosphate on recombinant sArr would have to be carried out to determine if this is a useful system.
Recombinant arrestins of species other than squid have been produced in E. coli cells using different procedures for production and purification. For example, the C- terminal truncated B-arrestins were expressed, without any fusion tags, by T7 RNA polymerase in E. coli BL21(DE3] cells. The cells were grown by fermentation in 10 L volumes, and the protein was purified by affinity chromatography with heparin sepharose
[Han, Gurevich et al. 2001). The need for such a large scale production is indicative of the majority of the protein being insoluble as I found for sArr. The production ofLimuIus arrestin was similar to the method I used here, using BL21(DE3) cells with 0.4 raMIPT G induction (Sineshchekova, Cardasis et al. 2004). However, the purification was significantly different. Instead of extracting arrestin from the supernatant of whole cell lysates, the authors isolated arrestin from the pellet by sonication in the presence of lysozyme, followed by purification with heparin sepharose. This second lysis in their protocol was intended to break inclusion bodies to release arrestin protein. However, it seems more likely that the second lysis may have released arrestin from cell membranes rather than inclusion bodies which are usually dissociated in the presence of detergents or chaotropic agents such as urea. These methods were not useful in improving release of sArr from insoluble fractions in my studies.
Arrestin production for the purpose of crystallization has been performed with arrestin purified from bovine eyes rather than using recombinant arrestin. Some studies have used recombinant mammalian arrestins in which all of the cysteine residues were changed to avoid disulfide bond formations and improve solubility (Hanson, Van Eps et al. 2007]. This approach could be used in the future with sArr to improve the solubility of the protein in the starting bacterial extract.
In summary, arrestins from various species are difficult proteins to produce in bacterial cells as most of the proteins are insoluble. To date the most successful approach has been to grow large volumes of bacteria to produce relatively small amounts of purified proteins, or to express modified arrestins to increase production of soluble protein.
4.2 Phosphorylation of sArr by SQRK
Previous work in our lab, using nsArr purified from squid eyes, reported that sArr is phosphorylated in a light- and SQRK-dependent manner (Mayeenuddin and Mitchell
2003; Swardfager and Mitchell 2007). A mechanism for sArr phosphorylation has been proposed. Light-activated rhodopsin recruits both SQRK and sArr to cell membrane to initiate receptor desensitization. Subsequent interactions between rhodopsin and SQRK may activate the latter. The activated SQRK then carries out dual phosphorylation of both rhodopsin and sArr, with phosphorylation of sArr stimulated by the presence of Ca2+
[Swardfager and Mitchell 2007). With enriched rsArrWT, I confirmed that the sArr phosphorylation was SQRK-dependent, and the phosphorylation level was significantly increased [by ~50 %) in the presence of membranes. However, in the absence of membranes, both native and recombinant sArr could be phosphorylated by SQRK and the phosphorylation level was not affected by the presence of Ca2+, suggesting that SQRK phosphorylation of sArr in the cytosol may be regulated differently than SQRK phosphorylation of sArr when both are attached to rhodopsin. The phosphorylation sites on sArr are Ser392 and/or Ser397 at the protein's C-terminus, however, we have not determined if one or both of these sites are phosphorylated.
4.2.1 Membrane Effect on sArr Phosphorylation Most of our previous phosphorylation assays were carried out in the presence of photoreceptor membranes, preventing analysis of any potential sArr modifications prior to its binding to membranes or rhodopsin. Since sArr is a soluble protein, any modifications of the protein in the cytosol may significantly alter its membrane recruitment and even receptor desensitization. The results of my study suggest that in addition to the membrane-dependent phosphorylation, a portion of sArr can be phosphorylated in the cytosol. In previous studies carried out using purified nsArr and
SQRK, significant phosphorylation of nsArr in the absence of membranes was not observed
[Swardfager and Mitchell 2007). This discrepancy is likely the result of different exposure times used in autoradiography. Since nsArr was strongly phosphorylated in the presence of membranes, short exposure times were sufficient to observe good phosphorylation, and nsArr phosphorylation in the absence of membranes was very low. In my studies I examined rsArrWT or nsArr phosphorylation without membranes and found that this could be seen with longer exposure times. Therefore it seems that SQRK can phosphorylate sArr in the cytosol but this is a relatively weak process compared to the phosphorylation on the membrane.
The cytosolic arrestin phosphorylation may not be unique for squid arrestin.
Limulus and Drosophila visual arrestins were phosphorylated in the dark prior to rhodopsin activation (Matsumoto, Kurien et al. 1994; Battelle, Andrews et al. 2000).
Surprisingly, almost 90 % of Limulus arrestins were phosphorylated in the dark at one or more of their three phosphorylation sites: Ser377, Ser381, and Ser396 (Battelle, Andrews et al.
2000). Both Ser377 and Ser381 were significantly phosphorylated in the dark, whereas
Ser396 was significantly phosphorylated only in the light (Sineshchekova, Cardasis et al.
2004). This differential phosphorylation results in multiple isoforms of Limulus arrestins. Since all 3 phosphorylation sites are within the C-terminal regulatory domain, each phosphorylated isoform may function differently in regulating rhodopsin function and signal transduction (Sineshchekova, Cardasis et al. 2004).
A sequential phosphorylation model may also apply to sArr, if phosphorylation occurs at both serine residues identified in my study. In a sequential phosphorylation model, sArr would be phosphorylated on one serine in the cytosol, and then on the other serine after being recruited to the membrane. This model may explain the significant increase in phosphorylation when membranes were added in my assays. I propose that the interaction between arrestin and metarhodopsin results in a conformational change in arrestin, which subsequently exposes both phosphorylation sites on sArr to SQRK. In the cytosol only one site may be available for SQRK phosphorylation when the C terminal tail of the molecule is embedded within the proteins core. A simpler explanation for the improved phosphorylation of sArr when bound to rhodopsin may be the improved interaction of sArr with SQRK also bound on rhodopsin. In the cytosol, the probability for the two soluble protein's interacting would be much lower than when both are in close proximity bound to rhodopsin. In future studies, the successful production of rsArr2Al
(S392A) and rsArr2A2 (S397A) proteins and their reconstitution with SQRK may provide the means to determine potential phosphorylation differences at the two sites.
4.2.2 Calcium Effect on sArr Phosphorylation
The evidence for calcium effects on SQRK-mediated phosphorylation has been reported in previous studies from our lab (Mayeenuddin and Mitchell 2003; Swardfager and Mitchell 2007). Stimulatory effects of Ca2+ were demonstrated on sArr phosphorylation in the presence of membranes in the light. In my study, using rsArrWT and nsArr, a stimulatory effect of Ca2+ in solution was not found, suggesting that the Ca2+- stimulated sArr phosphorylation may require SQRK and/or sArr binding to membranes or
rhodopsin. The Ca2+-effect is most likely mediated by SQRK which has two putative
Ca2+/calmodulin binding sites deduced from previous sequence analysis (Mayeenuddin
and Mitchell 2001). As previously suggested, the binding of SQRK to metarhodopsin may
change its conformation, which likely exposes its sites for Ca2+ binding (Swardfager and
Mitchell 2007). The increase of Ca2+ level, from 0.5 uM before light activation to 100 uM
after light activation (Rack, Xhonneux-Cremers et al. 1994), may facilitate the potential
Ca2+/calmodulin-mediated SQRK stimulation. The net result of such stimulation is
increased sArr phosphorylation. Moreover, this increase in phosphorylation seems to
require the conformational change of sArr induced by metarhodopsin. Future studies may
provide more evidence for the mechanism by which Ca2+/calmodulin modifies SQRK
interaction with its substrates.
4.2.3 Phosphorylation and Clathrin-Binding Sites on sArr
The phosphorylation sites of sArr are Ser392 and/or Ser397 at the protein's C-
terminus. Similar C-terminal phosphorylations have been reported in different arrestins
and associated with regulatory functions such as receptor internalization. Studies of
mammalian arrestins demonstrated that the phosphorylation at C-terminus inhibited
arrestin-mediated receptor internalization. Mammalian (3-arrestins have both the LIEF
binding motif for clathrin and the RXR binding motif for (32-adaptin at their C-terminus,
suggesting that the C-terminal phosphorylation may directly affect arrestin recruitment of
endocytic factors (Luttrell and Lefkowitz 2002). Therefore, removal of the
phosphorylation is required for receptor internalization. The RXR adaptin motif was also
found in the Limulus visual arrestin that has also been reported to internalize with rhodopsins (Sineshchekova, Cardasis etal. 2004). An amino acid sequence analysis of sArr has identified a potential clathrin binding motif- 374LIMEEF379 at the C-terminus. Future investigations of sArr-clathrin interaction maybe of interest.
4.3 Functional Role of the Phosphorylation
4.3.1 Phosphorylation and Arrestin Binding to Membranes
Squid photoreceptor rhabdomeric membranes are enriched with rhodopsin proteins. We demonstrated both recombinant and nsArr bound to the membranes in a light-dependent manner, suggesting that sArr can discriminate the active and inactive rhodopsin conformations and selectively bind to the latter.
The binding of metarhodopsin may occur in a sequential order as illustrated in
Figure 8. The initial interaction between arrestin and the intracellular loops of metarhodopsin may disrupt arrestin's polar core structure, which may then facilitate formation of a stable arrestin-metarhodopsin complex in the second step. The release of arrestin's C-terminus is also involved in the complex formation. The C-terminus of the arrestin is folded within the core of the molecule in the inactive state, and moves out of the core when the polar core is disrupted. Mutations from serines and threonine to alanines in sArr may have disrupted the interaction of the C-terminus with the polar core and in someway increased arrestin binding to metarhodopsin. However, the effect of these mutations must be explored further before we can understand their effects.
Phosphorylation of nArr had no effect on its binding to light-activated membranes.
However phosphorylation did inhibit nsArr binding to dark-adapted membranes. Given that phosphorylation of the C-terminal tail will add one or more additional charges to this area of the molecule, it is likely to inhibit C-terminal tail interaction with the polar core, and may therefore disrupt stable arrestin receptor binding. Thus allowing phosphorylated arrestin to dissociate from rhodopsin when it is isomerizes back to the dark state. To our knowledge this inhibitory effect has not been reported previously for other arresins, and may be of significance for arrestins that are phosphorylated in the absence of activated receptors, such as (3-arrestins, Drosophila, Limulus, and our squid arrestin. The percentage of soluble nsArr that was phosphorylated was likely small (as assessed by autoradiography) and therefore it is difficult to determine the significance of this finding at the present time. Homogeneous recombinant sArr preparations would be useful to clarify the extent of sArr phosphorylation and how the phosphorylation affects its binding to inactive rhodopsin.
In summary, my study has suggested that phosphorylation of arrestin inhibits its ability to bind to inactive rhodopsin but has no effect on its binding to metarhodopsin.
4.3.2 Phosphorylation and Arrestin Dissociation from Membranes
Although invertebrate metarhodopsin and rhodopsin are photoconvertible, the difference in absorbance maxima between the two is only 7 nm, which is beyond our ability to manipulate in the lab. Therefore, we used dark adaption as a means of converting metarhodopsin to rhodopsin. Our previous study using purified native sArr found that in the presence of high salt concentrations, sArr can be effectively dissociated from membranes in the dark (Swardfager and Mitchell 2007). The increased ionic strength that disrupts the sArr-rhodopsin complex has very little effect on the sArr-metarhodopsin binding, suggesting that interaction between arrestin and metarhodopsin is of very high affinity. In this thesis I took advantage of the effect of ionic strength in the buffer to test the apparent affinity of phosphorylated and unphosphorylated sArr in dissociation assays from dark-adapted retinal membranes. The lower affinity of phosphorylated sArr for binding to rhodopsin was reflected in its dissociation at a lower ionic strength, 584 mM compared to 883 mM NaCl for 50% dissociation of unphosphorylated sArr. The effect of phosphorylation on arrestin dissociation from rhodopsin has also been observed in the
Drosophila visual system, in which only the phosphorylated arrestin could dissociate from
inactive rhodopsin. A mutation at the phosphorylation site in Drosophila arrestin resulted
in the formation of an abnormally stable arrestin-rhodopsin complex (Alloway and Dolph
1999; Alloway, Howard et al. 2000). These findings have demonstrated that the
phosphorylation of arrestin plays an important role in receptor dissociation.
In invertebrate arrestins, the lower binding stability of phosphorylated arrestin
may be the result of charge repulsion from the phosphate groups. We know from the
crystal structure of mammalian arrestins that the C-terminus undergoes the greatest
conformational change upon binding to receptors, it is folded into the protein core in the
protein's inactive state and moves out of the core upon receptor binding. Therefore,
phosphorylation on the C-terminus of arrestin and also on rhodopsin would add additional ionic charges to their interaction site and allow arrestin to more readily dissociate from rhodopsin once it has been photo-converted to its inactive state.
4.4 Modeling the Arrestin-Mediated Receptor Desensitization and Future Studies
In summary, my findings have determined the phosphorylation sites of squid arrestin at Ser392 and/or Ser397 at the protein's C-terminus. The squid arrestin can be phosphorylated in the absence of light-activated rhodopsin and this may help prevent arrestin binding to inactive rhodopsin. The light-activated rhodopsin recruits both SQRK and arrestin to the membrane, resulting first in receptor desensitization and then in receptor phosphorylation. After binding to metarhodopsin, arrestin hinders iGqoc activation by metarhodopsin, resulting in the termination of phototransduction. Arrestin binding to metarhodopsin also increases its phosphorylation by SQRK, possibly by bringing the two proteins into close proximity and rendering SQRK sensitive to calcium activation. As the metarhodopsin is photoconverted back to rhodopsin, phosphorylated arrestin dissociates to facilitate the reuse of rhodopsin. If metarhodopsin is not converted back to rhodopsin the bound arrestin may recruit endocytic factors such as clathrin to its
C-terminal clathrin-binding site and initiate internalization of the arrestin-rhodopsin complex (Figure 26].
To complete the studies of our model, two main directions should be considered in future studies. The first is to find the phosphatase responsible for dephophorylation of rhodopsin and arrestin. Purification and cloning of this phosphatase would allow us to test the role of dephosphorylation in resetting the components of the invertebrate visual system back to the dark state. The second direction that could be pursued is to determine if sArr does bind to clathrin and if the identified LIMEEF sequence is the clathrin-binding site. This may provide a molecular mechanism for the process of receptor internalization that has been demonstrated in other invertebrate visual systems. Future studies should also clarify the phosphorylation at Ser392 and Ser397, which may determine if sArr is phosphorylated in a site-dependent manner.
During my studies I also tested the expression of sArr in the human embryonic kidney (HEK293) cell line. sArr was expressed in the cells, and although this was not pursued, this model offers an additional avenue for testing the effect of arrestin modifications on its ability to associate and dissociate from activated G protein-coupled receptors. Future experiments with sArr stably transfected into HEK cells could be tried using wild type sArr as well as mutant sArr constructs in which Ser392 and Ser397 are replaced with aspartic acid residues to simulate phosphorylation. A similar approach was taken with (3-arrestin to demonstrate the effect of phosphorylation on arrestin-mediated receptor internalization (Lin, Krueger et al. 1997]. Rhodopsin Metarhodopsin 493 nm /V Arrestin Phosphatase
> Arrestin Dephosphorylation & Dissociation Metarhodopsin + > Rhodopsin Phosphorylated Phosphorylated / Dephosphorylation Rhodopsin Unphospohrylated > Translocation Arrestin
500 nm Phosphorylated SQRK Arrestin Dissociation V
Phosphorylated Phosphorylated Metarhodopsin C Metarhodopsin + Unphosphorylated Clathrin + Phosphorylated Arrestin Arrestin
Figure 26. Proposed Model of Rhodopsin Biochemical Cycle in Invertebrate Photoreceptors. Activation and inactivation lights represents different light wavelengths that are responsible for activating and inactivating invertebrate rhodopsin, respectively. 5. Appendix 1. Construction of recombinant protein expression vectors rsArr-pDEST17.
sArr cDNA
2. Construction of recombinant protein expression vectors rsArr-pDEST15.
RBS I ATG GST sArr cDNA
3. Construction of recombinant protein expression vectors rsArr-pDESTHisMBP.
ATG 6xHis MBP sArrcDNA TTtc™
72 6. Reference
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