Biochimie ( 199 i ) 73, 109-120 © Soci6t6 franqaise de biochimie et biologie moi6culaire / Elsevier, Paris

109

Towards understanding the glycoprotein hormone receptors R S a l e s s e , JJ R e m y , J M L e v i n , B J a l l a l * , J G a m i e r * * Unit~ d'Ing~nieHe des Protdines. INRA-Biotechnologies, 78352 Jouy-en-Josas Cedex. France

(Received 3 October 1990; accepted 23 November 1990)

Summary - - Lutropin (LH), tollitropin (FSH) and thyrotropin (TSH), as well as choriogonadotropin (CG, which binds to the LH receptor) constitute the glycoprotein hormone family. Their 3 receptors have been cloned during the last few months. They belong to the large group of G-protein coupled membrane proteins, with their specific N-terminal domain likely to bind the hormone and the characteristic 7 membrane-spanning segments in their C-terminal moiety. The present review discusses the main results of amino acid sequence analysis performed on the glycoprotein hormone receptors. The putative extracellular head exhibits < 45% homology over the 3 receptors, while = 70% residue conservation is found in the transmembrane moiety. Here only, limited sequence homologies (= 20%) can be found with other G-protein coupled receptors. The secondary structure predictions performed on the 3 receptors revealed that the polypeptide sequence predicted as ordered (either t~-helix or B-strand) were repeated evenly throughout the extracellular head with a period of = 25 amino acids. This analysis helped to define the intervening loops between this ordered stretches as potential candidates for bearing at least part of the binding site of the hormones. Some of the perspectives opened by the cloning of the receptors are described, like the production of the extracellular head of the porcine LH receptor in baculovirus-infected insect ceils, and the exploration of the LH receptor's mechanism of functioning as a dimer. glycoprotein hormone receptors / sequence analysis / secondary structure prediction / recombinant protein / expression / ligand blotting

Introduction Sixteen years elapsed between the first reported attempt to purify the LH receptor [1] and its simultaneous cloning in the rat [2] and in the pig [3]. Noticeably, both results were obtained through the alliance of one molecular biology group and one gonadotropin group. Eight months later, all the members of the glycoprotein hormone receptor family, namely the TSH receptor, either canine [4], human [5, 6, 7] or murine [8] and the rat FSH receptor [9] had been cloned and their c D N A sequenced. This is thus a good opportunity to review past results and *Present address: Max Planck Institiit fiir Biochemie, Dept Molecular Biology, Am Klopferspitz, 18A, 8033-Martinsried, Germany **Correspondence and reprints Abbreviations: LH, iutropin or luteinizing hormone; FSH, follitropin o,," follicle-stimulating hormone; TSH, thyrotropin or thyreo-stimulating hormone; CG, choriogonadotropin; N-domain, N-terminal moiety of the glycoprotein hormone receptors (residues 1-399); TM-domain, transmembrane domain (residues 400-662); C-domain, C-terminal (663-end); SDS, sodium docecyl sulfate; PAGE, polyacrylamide gel electrophoresis

future prospects in the domain hormone receptor research.

of

glycoprotein

The glycoprotein h o r m o n e family In mammals, the glycoprotein hormone family comprises 4 hormones, which bind to 3 high affinity receptors. There are 3 gonadotropins: lutropin (luteinizing hormone, LH), follitropin (follicle stimulating hormone, FSH) and choriogonadotropin (CG, a LHlike hormone, but of placental origin and which has been found only in primates and equines) and one thyrotropin (thyroid stimulating hormone, TSH). These hormones are composed of 2 dissimilar glycoprotein subunits, namely ~ and I~, whose association is mandatory for expression of biological activity and specificity [10]. Nevertheless, in a given species, the t~ subunit is identical in all the hormones and the different B subunits present large sequence homologies with close secondary and tertiary structures [ 11 ]. This suggested an evolutionary overview of this family [ 12]. As such similarities were found between the hormones, it was expected that their receptors would

! 10

R Salessc et a/

Overall topology of the receptors

display, by parallel evolution, largely similar seque{lce and structural features: a model of binding ~as proposed, named "negative specilicity', which predicted that the high binding affinity of the receptors was due to common structural regions both in the hormones and in the receptors, but that some discriminating motil/s would prevent cross-binding 1131. Moreover, as common epitopes had been found between the LH and the TSH receptors 1141, it was expected that the cloning of one receptor of the family would open the way lor the cloning of the others, which was true for TSH receptors 15, 71 and for the FSH receptor 191.

All the receptors can be considered as composed of a putative bulky extracellular N-terminal domain up to residue 399 (N-domain) followed by a transmembrane domain (residues 400-662) with 7 transmembrane fragments (TM-domain) and by a short, presumably cytoplasmic, C-terminal domain (C-domain) (fig 2). This overall topology could be expected on the basis of their known adenylyl cyclase coupling. However, glycoprotein hormone receptors exhibit only a weak homology with the G-protein coupled receptors, and which is limited to the TM-domain (23%, 131). The glycoprotein hormone receptors differ in the importance of their N-domain (350--400 residues) as compared to a few tenths residues in the other receptors. Such a topology was previously supported by immunochemical 116, 171 and proteolytic studies 118-211, as well as chemical cross-linking experiments 122]. Antibodies against N-domain peptides recognized the receptor in intact cells, but these cells must be permeabilized to be labeled with antibodies against the C-domain peptides [231.

Sequence analysis of the receptors Figure I displays the sequences of the 6 receptors cloned to date, which were aligned with a multiple sequence alignment algorithm 1151. The following text will take into account the amino acid numbering of this figure, including the presence of gaps. Table I depicts the sequence identities in more detail: in the rat, where the 3 receptors have been cloned, the overall sequence homology between the receptor is < 55%; however, in the transmembrane region, it increases to = 70%. It falls to < 45% in the N-domain, and it is very low in the N-terminal tail (= 25%).

The N - d o m a i n

Here, no homology can be found between the receptors and other G-protein coupled receptors. We first notice that the TSH receptors exhibit a 52-amioo acid insertion between residues 306 and 359, and the FSH

Table I. Identical residues in the different regions of the receptors. Comparison of the amino acid sequences of the glycoprorein hormone receptors. The table compares the rat (upper part), LH (middle) and TSH receptors (lower part). The sequences have been divided into the N-domain (!-399), TM-domain (400--622) and C-domain (see text). The number of identical amino . . . . . . . . •..,,.,, ,.,o,,,.,,,, ,=, ,,,~..,~,,.~ ,,, ,,,~. ,,.,~,,., ,~.,, part u, ,.,~,.,, ,,,,,,,,^. ,~,,,~ ,,,,.,, pcrccntagc over . . . . . . . . . . . . . . . . . . . . . Ui~F,,~, ,E, t t t part of each matrix. Calculations were performed only over the polypeptide segments common to all receptors (gaps ignored). ....

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Fig 1. Sequence comparison between the glycoprotein hormone receptors. The sequences were aligned according to the multiple alignment program of Levin [ ! 5]. Boxes of conserved matches are drawn. Hydrophobic replacements of hydrophobic residues, charge conservation or aromatic residues interchange were considered as conservative, pLH-R and rLH-R: porcine and rat LH receptors; cTSH-R, hTSH-R and rTSH-R: canine, human and rat TSH receptors, rFSH-R: rat FSH receptor.

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Fig 2. Overall topology of the porcine LH receptor. The amino acid sequence was taken from [3]. The transmembrane segments were drawn on the basis of sequence homologies with other G-protein coupled receptors. Filled circles = Cys residues; potentially glycosylated Asn are indicated by Y.

Glycoprotein hormone receptors receptor a 7 amino acid insertion at the same place as compared to the LH one (fig 1). This variable region could be an adaptative segment between the hormonebinding domain and the TM-domain, which in mammalian B-adrenergic receptors stems from intronless genes [24]. The reported alternative splicing of the cDNA, which takes place at Leu 299 in porcine [3] and rat [25] LH receptors, reinforces this view. A secondary structure prediction was made with the program Combine [26]. As seen in figure 3, predicted periodic secondary structures (either t~-helix or B-strand) display some periodicity along the receptor sequences. Secondary structure predictions have some limits: for instance, B-strands are generally underpredicted; the predicted boundaries of fragments with periodic structures are not always clear [26]. The secondary structure prediction was thus analysed from a broad point of view taking into account only the periodic stretches common to all sequences which are boxed in figure 3. Figure 4 shows that some of these periodic stretches were found at a mean distance of - 23-27 amino acids from each other, and others at a distance of around 8-12 amino acids. The distance of 23-27 correlates with the length of the 14 imperfectly repeated motifs detected in rat LH [2] and FSH [9] receptors. The interest of our analysis was to forecast that some of the loops intervening between stretches of periodic structure could constitute at least in part the region of hormone-receptor interaction. Table II displays the ~egions possibly present as loops and some of their characteristics. The loops exhibiting more conservation and hydrophilicity may be considered n ~ n n r l D f t h e h l n d l n a c i t e c ' a m m a n t n ~11 t h . hormones, whde less conserved ones may h~ve a discriminating role among the hormones. ~,-vv . . . . . . . . . . j information concerning exposure of some regions to the solvent come from the fact that loops 44--56, 73-83, 144-158, 171-181, 250-274, 277-300 bear potentially glycosylated Asn. Since deglycosylation of the LH receptor [27] decreases its apparent molecular weight by 28 kDa, it is reasonable to assume that at least 2 or 3 Asn are glycosylated. The same holds true for the TSH receptor on the basis of c D N A expression experiments [28]. One intriguing result of LH receptor cloning was the finding of an 1 l-amino acid peptide (249-260, [2]) homologous to a segment in soybean lectin [29], but variable among the glycoprotein hormone receptors. As the sugar moiety of the glycoprotein hormones seems to play an important role in hormonal stimulation [30], it was tempting to assign a role in sugar recognition to this region of the receptor, inasmuch as the above sequence analysis indicated this loop (250-261) as a candidate for hormone binding. However, the amino acids participating in the binding

113

Table II. Characteristics of the predicted loops in the glycoprotein hormone receptors. The loops were defined as the intervening sequences between the boxes defined in figure 3. As some of the boxes are short (1 amino acid), some loops (displayed between brackets) were extended in the N- or C-terminal direction. Loops bearing a conserved potentially glycosylated Asn are indicated by an *. Loop location

Length

18-31 a

14

1

44-56 (or 37-56)

13 (20)

5 (7)

(+)

3 (4)

96-106 (or 87-106)

1i (20)

6 (7)

+ (+ +)

4 (6)

113-123 (or 113-129)

11 (17)

7 (10)

(+)

1 (3)

144-158 (or 137-158)

15 (22)

7 (10)

(--)

1 (4)

171-181 (or164-181)

11 (18)

7* (11")

4 (9)

198-208

11

6

4

212-224

13

7

7

250-261 264-274 (or250-274)

12 11 (25)

8 4 (14)

277-289

13

2*

Number of Conserved Conserved conservative charges hydrophilic matches residues

+ (+)

6 3 (9) 2

alt has been reported that deletion or substitution of the i 522 sequence in the TSH receptor abolished TSM binding [Wadsworth HL, Chazenbalk GD, Nagayama Y, Russo D, Rapoport B (1990) An insertion in the human thyrotropin receptor critical for high affinity hormone binding. Science 249, 1423-1425]

of the sugars in the parent lectin concanavalin A [31] do not belong to the above sequence, which reduces its interest. Other residues which deserve attention are the cysteines. With 12 such residues in its N-domain, the porcine LH receptor appears rather richly endowed, but there is no cysteine-rich region as defined in the low density lipoprotein receptor, for instance [31a]. Only 6 homologous Cys in the 3 sequences (fig 4) can be found: Cys 8, Cys 262-263, Cys 371,379 and 389. As they are in even number, they could be involved in 3 disulfide bridges.

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Fig 3. Secondary structure prediction on glycoprotein hormone receptors. The prediction was performed with the Combine pro~am [26]. Sequences predicted in et-helix or B-strand in the 3 receptors are simultaneously boxed. H: o~-helix; E: extended (B-strand); no letter: residues predicted in aperiodic structure (random coil). The imperfect repeated motifs, derived from leucine-rich adhesion proteins as described in McFarland et al [21 in the N-domain are delimited by arrows and numbered in arabic numerals. The putative transmembrane segments are delimited by square brackets and numbered in roman numerals. N: potentially glycosylated Asn; + and -: conserved charged residues.

Glycoprotein hormone receptors

3

conserved (623), and the second conserved or replaced by a Tyr. This suggests that the mechanism of hormonal transduction of retinal to rhodopsin or of catecholamines to the adrenergic receptor may be lost in the glycoprotein hormone receptors.

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Distance between periodic stretches (number of residues)

Fig 4. Periodic secondary structure frequency along receptors sequences. Figure 3 served as a basis for this analysis. The distance (in amino-acid residues) between the center of successive boxed sequences (predicted as periodic) was measured. Distance values were subdivided in 7 groups (4 residue long) as indicated along the abscissa axis. The height of every stack portion corresponds to the length of every boxed sequence. The numbers ill every stack portion correspond to their location in the repeated motifs delimited by arrows in figure 3.

The TM-domain Among the features that deserve attention one observes that the putative transmembrane segments are all mainly predicted as o~-helix (fig 3), which correlates with experimental results obtained with bacteriorhodopsin [32] and rhodopsin [33]. In these proteins, the 7 transmembrane helices appear to delimit a barrel-like transmembrane cylinder, with the apolar face of the helices turned toward the membrane lipids [34]. Noticeably, Cys 475 and 550 which form a disulfide bridge in the f~-adrenergic receptor [35] are present. Figure 5 shows that most of the amino acids implicated in the binding of either retinal or catecholamines [36] have disappeared in the glycoprotein hormone receptors: the Asp residue (113 in the B-adrenergic receptor [37]) is replaced by a Thr at position 482 (3rd transmembrane segment), 2 Ser (204 and 207 in the fifth transmembrane segment of the 8-adrenergic receptor [38]) are replaced at positions 567-568 by 2 hydrophobic residues. In the seventh transmembrane segment, at around position 649, the glycoprotein hormone receptors do not have the counterpart to Lys 296 in rhodopsin, where retinal is covalently attached [33] and where 13-adrenergic receptor bears a Trp (313) which is probably implicated in catecholamine binding [39]. Of the 2 Phe residues [38] at positions 289-290 in the 8-adrenergic receptor, the first is

The first cytoplasmic loop is suspected of being responsible for the correct membrane insertion of the B-adrenergic receptor [40]. Although little sequence homology exists between glycoprotein hormone receptors and the other G-protein coupled receptors, the presence of a positive charge (Lys 426) close to a conserved Leu (Leu 427) appears sufficient for a corrc,,t insertion. The third cytoplasmic loop has retained the attention of researchers working on the B-adrenergic receptor. Site-directed mutagenesis has indicated that the 2 peptides corresponding to positions 583-591 and 598-606 in the glycoprotein hormone r,:ceptors have importance in the coupling with the G-proteins [38]. In particular, the deletion of the 583-591 peptide abolishes both ligand binding and hormonal stimulation [40].The presence of a highly variable region between these 2 segments does not seem to be important [39]. Rather, positively charged residues at the cytoplasmic end of the 5th and 6th transmembrane segments (in particular the highly conserved Lys 602) appear as determinant in G-protein stimulation [40]. Cys 617, which is common to all G-protein coupled receptors inclusive of the glycoprotein hormone receptors, also plays a role in adenylate cyclase stimulation [35]. The C-domain of the B-adrenergic receptor has also been strongly implicated in the coupling to Gs. Here too, the presence of positively charged amino acids seems to be important for G-protein stimulation, with the conserved Arg or Lys at position 667 or 668 [40]. One interesting peptide capable of mimicking G-protein stimulation by natural receptors is the bee venom mastoparan. At the interface between a biological membrane and the solvent, this peptide adopts a helical conformation with its hydrophobic residues facing the membrane and 4 positively charged amines towards the solvent [41]. Mastoparan could mimick in some way the cytoplasmic loops of the B-adrenergic receptor [39]. In figure 3, it can be seen that the 2 cytoplasmic (but close to the membrane) segments 599-607 and 663-674 meet the appropriate requirements to function like mastoparan, with their high positive charge and predicted at-helix secondary structure. Taken together, all these features of the glycoprotein hormone receptors make them bona fide members of G-protein coupled receptor family. Recent experimental evidences essentially confirm this analysis [Chazenbalk GD, Nagayama Y,

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Fig 5. Comparison of the transmembrane region of several G-protein coupled receptors. The figure compares the sequences of 3 glycoprotein hormone receptors and those of rhodopsin (A, [54]) and B2-adrenergic receptor (B, [55]). The putative transmembrane segments are numbered in roman numerals a,d were based on those proposed for rhodopsin and B2-adrenergic receptor• Conserved sequences, as defined in figure I, are boxed. Conserved charges are indicated. Gaps in the sequence of glycoprotei_n_ ho__rrn_onerecepmr~ are indientod hy ~ Th,~ nhncnhnt~,l,~t,aiMa q,=."1,,~eh,~~ ,,~;.-,,I tail ^" D . . . . . . a--,:----~ .... ,, as the 2 Cys (675-676) which are palmytoylated at positions 322 and 323 in rhodopsin [56]. . . . . . . . . . . . . . . . . . . .

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Russo D, Wadsworth HL, Rapoport B (1990) Functional analysis of the cytoplasmic domains of the human thyrotropin receptor by site-directed mutagenesis. J Biol Chem 265, 20970--20975]. The regulation of B-adrenergic receptor activity by phosphorylation has been amply documented and appears dependent on the presence of a specific kinase (Bar-kinase [421. Although there have been reports showing in vitro phosphorylation of the rat LH receptor [43], the C-domain of the glycoprotein hormone receptors is much less rich in potentially phosphorylatable Ser or Thr than the catecholamine receptor (fig 5). In the porcine LH receptor, only Ser 706 and Thr 713 could be a target for protein kinase C phosphorylation [3]. This does not completely rule out a regulation of the LH receptor activity by phosphorylation, as only one phosphorylation site is sufficient for modulating the activity of receptors such as the

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insulin one [44], but limits its effect as compared to the B-adrenergic receptor. Perspectives

The availability of the c D N A of the glycoprotein hormone receptors now opens the way to the resolution of some problems raised in their study. Here, we have chosen to address only 2 questions: the production of the recombinant protein and the mechanism of transduction of the LH signal in cells naturally expressing the LH receptor. Production o f recombinant receptors In order to obtain the large amounts of pLH receptor necessary for its physical and structural analysis, we

Glycoprotein hormone receptors have chosen the baculovirus eucaryotic expression system [45]. Assuming that the N-domain of the receptor represents the extracellular and soluble binding site as reported in previous studies [46] and recently confirmed experimentally by two groups [Tsai-Morris CH, Buczko E, Wang W, Dufau ML (1990) lntronic nature of the rat luteinizing hormone receptor gene defines a soluble receptor subspecies with hormone binding activity. J Biol Chem 265, 19385-19388; Xie YB, Wang H, Segaloff DL (1990) Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity. J Biol Chem 265, 21411-21414], we have introduced 2 different cDNAs encoding this ectodomain into the unique BamHi cloning site of the transfer vector pVL941, pVL941 is a baculovirus-derived vector including the polyhedrin promoter, parts of the polyhedrin coding region, the 5' end of the polyhedrin gene being mutated at the initiation codon from ATG to ATT, so that translation should begin at the initiator of the foreign cDNA and expression result in the synthesis of the native (unfused) foreign protein [47]. Both constructs, pVLHR1 and pVLHR2, include the initiating codon and the signal peptide of the native molecule. They differ in their 3' region, one being stopped at the Phe 280, and the other at the Ash 406 residue. Following cotransfection of SF9 insect cells with the transfer vectnrs and the baculovirus AcNPV DNA, selection of positive recombinant viruses was performed by serial dilution and dotblotting with a pLH receptor probe. Then 3 rounds of plaque purification led to the selection of several pure recombinant viruses without occlusions for either In order to analyse the expression of recombinant proteins, SF9 cells were infected with either recombinant or wild-type baculoviruses, Coomassie blue stained electrophoresis (fig 6) revealed that, as compared to control non-infected cells, polyhedrin (32 kDa) is the most abundant product in wild-type AcPNV infected cells, while cells infected with the recombinant viruses express, at a very high level, proteins with apparent molecular weights of 45 kDa and 58 kDa respectively corresponding to construct pVLHRI and pVLHR2. Time-course expression of these recombinant products is comparable to polyhedrin, which is expressed steadily from the first day until at least the fifth day following infection (data not shown). These apparent molecular weights are consistent with the expected glycosylated forms of the 2 pLH receptor constructs. Moreover, as shown in figure 7, both proteins are specifically recognized by a monoclonal antibody directed against the extra-cellular part of the receptor [47a], when analyzed by

! 17

western blotting. Work is in progress to determine if this shortened form of the pLH receptor could bind the hormone with a high affinity. ls dimerization of the LH receptor implicated ill its .functioning? As dimerization of some receptors, including the epidermal growth factor receptor [481 and the transferrin receptor [49], seems to play a role in their activation, it was interesting to test such a hypothesis for the LH receptor in Leydig cells. In solubilized pLH receptor preparations, we detected by ligand blotting experiments with [125I]hCG as tracer the

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The aim of this review was to point out a few aspects of glycoprotein hormone receptor structure and function. The cloning of these receptors opens the

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446

f dimeric form (180 kDa) as the main product, with traces of the monomer (fig 8). This agrees with the apparent molecular weight of the pLH receptor detected in sucrose gradient centrifugation experiments [47al. Using ligand blotting, the monomeric form (90 kDa) has been found in Triton extracts or purified preparation of the rat LH receptor [16, 501. However, it has been demonstrated that the amount of dimeric form is decreased in the presence of N-ethvlmaleimide, which blocks disulfide bridge formation between 2 receptor molecules 1511. This could be relevant to receptor functioning, as a high transthiolase activity of LH and FSH has been reported [52]. which could thus modulate the state of receptor aggregation upon binding. Further experiments should be performed to determine whether such a dimerization occurs in vivo or with transfected cells upon hormonal stimulation.

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Fig 8. Revelation of the LH/hCG receptor by ligand blotting. The receptor was solubilized in 0.8% Triton X-100 from testis homogenate. 100 !.11 of the solution (approx 0.8 pmol) was subjected to electrophoresis. The gel was then electrotransferred onto nitrocellulose as described [ 16] and the receptor revealed with [1251]hCG in the absence (lane 1) or presence (lane 2) of an excess (10 lag) cold hCG.

Glycoprotchi hormone receptol.,, w a y ['or f u r t h e r studies c o n c e r n i n g s t r u c t t t r e - f u n c t i o n r e l a t i o n s h i p s , i n c l u d i n g s i t e - d i r e c t e d m u t a g e n e s i s , and the r e g u l a t i o n o f t h e i r e x p r e s s i o n in cells s e n s t t i v e to g l y c o p r o t e i n h o r m o n e s . As these cells are p r e s e n t in o n l y a l i m i t e d n u m b e r o f target tissues, this m a k e s t h e m s u i t a b l e m o d e l s o f d i f f e r e n t i a t i o n [53 !.

Acknowledgments We acknowledge the skilful technical assistance of Denise Grebert. This work way financed in part by the Fondation pour la Recherche Mddicale and grants from the Ministbre de la Recherche et de la Technologic and from the lnstitut National de la Recherche Agronomique. We thank Misa for her share in typing and verifying the anaino acid sequences.

12

13 14

15 16

17

References I 2

3

4

5

6

7

8

9

10 !1

Dufau ML, Cart KJ (1973) Extraction of soluble gonadotrophin receptors from rat testis. Nature 242, 246-248 McFarland KC, Sprengei R, Philipps HS, K6hler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH (1989) Lutropin-choriogonadotropin receptor: an ususual member of the G protein-coupled receptor family. Science 245,494-499 Loosfelt H, Misrahi M, Atger M, Salesse R, Vu Hai" MT, Jolivet A, Guiochon-Mantcl A, Sar S. Jallal B, Gamier J, Milgrom E (1989) Cloning and sequencing of porcine LHhCG receptor eDNA: variants lacking transmembrane domain. Sciem'e 245,525-528 Parmentier M, Libert F, Maenhau~ C, Lefort A, G6rard C, Perret J, Van Sande J, Dumont JE, Vassart G (1989) Molecular cloning of the thyrotropin receptor. Sciem'e 246, ! 620-1622 Libert F, Lefort A, Gdrard C. Parmcntier M, Perret J, Ludgate M, Dumont JE, Vassart G (1989) Cloning, sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding o!" au,,mntibodie.,-,. Bio~'hem Biophys Res Conmmn 165. 125(b- 1255 Nagayama Y. Kaufman KD. Seto P. Rapoport B (1989) Molecular cloning, sequence and functional expression of the eDNA for the human thyrotropin receptor. Biochem Biophys Res Commtm 165, I 184- i 190 Misrahi M, Loosfelt H, Atger M, Sat S, Guiochon-Mantel A, Milgrom E (1990) Cloning, sequencing and expression of human TSH receptor. Biochem Biophys Res COllllIIIlll ! 66, 394--403 Akamizu T, lkuyama S, Saji M, Kosugi S, Kozak C, McBride OW, Kohn LD (1990) Cloning, chromosomal assignment, and regulation of the rat thyrotropin receptor: expression of the gene ix regulated by thyrotropin, agents that increase cAMP levels, and thyroid autoantibodies. Proc Natl Acad Sci USA 87, 5677-568 I Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH (1990) The testicular receptor for follicle stimulating hormone: structure and functional expression of cloned cDNA. Mol Endocrinoi 4, 525-530 Parsons TF, Strickland TW, Pierce JG (1985): Disassembly and assembly of glycoprotein hormones. Methods Enzvmol i 09, 736-749 Gamier J (1084) l~tude comparative de I'hormone choriogonadotrope humaine et des autres hormones glycoprotdiques. Ann EndocHno145, 243-255

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129 31

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41

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45

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48

49

50 51 52 53 54 55

56

Smith GE, Fraser MJ, Summers MD (1983) Molecular engineering of the Autographa californica nuclear polyhedrosis virus genome: deletion mutations within the polyhedrin gene. J Viro146, 584-593 Kolena J, Seb/Skovh E (1986) Porcine foilicular fluid containing water-soluble LH/hCG receptor. Arch lnt Physiol Biochim 94, 1-10 Luckow VA, Summers MD (1988) Trends in the development of baculovirus expression vectors. Biotechnology 6, 47-55 Vu Hai Luu-Thi M, Jolivet A, Jailal B, Salesse R, Bidart JM, Houiller A, Guiochon-Mantel A, Gamier J, Milgrom E (1990) Monoclonai antibodies against luteinizing hormone receptor. Immuno-chemical characterization of the receptor. Endocrinology 127, 2090-2098 Cochet C, Kashles O, Chambaz EM, Borrello I, King CR, Schlessinger J (1988) Demonstration of epidermal growth factor-induced receptor dimerization in living cells using a chemical covalent cross-linking agent. J Biol Chem 263, 3290-3295 Alvarez E, Giror~s N, Davis RJ (1989) Intermolecular disulfide bonds are not required for the expression of the dimeric state and functional activity of the transferrin receptor. EMBO J 8, 2231-2240 Kein~inen KP, Kellokumpu S, Rajaniemi HJ (1987) Visualization of the rat ovarian lutropin receptor by ligand blotting. Mol Cell EndocHno149, 33-38 Sojar HT, Bahl OP (1989) Characterization of rat ovarian lutropin receptor. Role of thiol groups in receptor association. J Biol Chem 264. 2552-2559 Boniface JJ, Reichert LE Jr (1989) Evidence for a novel thioredoxin-like catalytic property of gonadotropin hormones. Science 247, 61-64 Amsterdam A, Rotmensch S (1987) Structure-function relationships during granulosa cell differentiation. Endocr Rev 8. 309-337 Ovchinnikov YA (1982) Rhodopsin and bacteriorhodopsin: structure-function relationships. FEBS Let! 148, 179-191 Dixon RAF, Kobilka BK, Strader DJ, benovic JL, Dohlman HG, Fr|e!le T, Bolanowski MA, Bennett CD, Rands E. Diehl RE, Mumford RA, Slater EE, Sigal IS, Caron MG, Lefkowitz RJ, Strader CD (1986) Cloning of the gene and cDNA for mammalian B-adrenergic receptor and homology with rhodopsin. Nature 321,78-79 K6nig B, Arendt A, McDowell JH, Kahlert M Hargrave PA, Hofmann KP (1989) Three cytoplasmic loops of rhodepsin interact with transducin. Proc Natl Acad Sci USA 86, 6878-6882