Exp. Clin. Endocrinol. loo (1992) 4-8

Experimental and Clinical Endocrinology © 1992 Johann Ambrosius Barth

Properties of Glycoprotein Hormone Receptors and Post-Receptor Mechanisms Wolfgang E. Merz

Key words: LH/hCG receptors - FSH receptor - Review Zusammenfassung: In den letzten Jahren ist die Aufklärung der Aminosäure-Sequenzen der Rezeptoren für die Glykoproteinhormone (Lutropin (LH), Follitropin (FSH), Thyrotropin (TSH) und Choriogonadotropin (hCG)) auf der Basis der Isolierung und Entschlüsselung der cDNAs gelungen. Erwartungsge-

sche Strukturen ausbilden können; sie sind vermutlich an der Ausbildung von Protein-Protein-Wechselwirkungen maßgeblich beteiligt. Die Transmembranregion der Rezeptoren besteht aus 7 Helices, die durch 3 extrazelluläre und 3 intrazelluläre Schlei-

fen der Polypeptidkette miteinander verbunden sind. Die 3.

Glykoproteinen bekannt war, ausgeprägte Honiologien in den Primärstrukturen. Die Rezeptoren gehören zu einer Superfamilie von G-Protein-bindenden Transmembranproteinen, zu denen auch das Rhodopsin und der 3-adrenerge Rezeptortyp zählt. Die

intrazelluläre Schleife scheint an der Kopplung der Rezeptoren an das intrazelluläre Signalsystem (über ein stimulierendes GProtein) beteiligt zu sein. Die Kontaktstellen der Glykoproteinhormone mit den Rezeptoren sind bislang ungenügend aufgeklärt. Insbesondere die Rolle des Kohlenhydratteils der Hormone bei der Rezeptorbindung und die Aktivierung des Signalkomplexes ist unklar. Die deglykosylierten Hormone binden

Glykoproteinhormonrezeptoren zeichnen sich durch eine beson-

verstärkt an die Rezeptoren, sie wirken aber teilweise als

ders große extrazelluläre Bindedomäne für die Liganden aus. Sie enthält 14 Leucin-reiche Sequenzmotive mit hochgradig

Antagonisten für die nativen Hormone. Eine direkte Beteiligung der Kohlenhydratteile an der Aktivierung der Signalkomplexe erscheint aber unwahrscheinlich.

mäß zeigen die Rezeptoren, wie dies auch schon von den

konservierten Konsensussequenzen, die vermutlich amphipathi-

Introduction The glycoprotein hormones thyrotropin (TSH), follitropin (FSH), lutropin (LH) and human choriogonadotropin (hCG) are members of a superfamily of relatively large hormone molecules (apparent molecular weights between 26-38 kDa). They are composed of two non-covalently linked dissimilar subunits, a common ce-subunit and 13subunits which are specific for each member. The hCG(3-subunit is discerned from the other 3-subunits by a Cterminal extension of 30 amino acids, possibly by a loss of the stop codon. The homology in the amino-terminal amino acid sequences of the (3-subunits (e.g. 80% in case of hLH-(3/hCG-13) as well as functional cross-reactivities based on receptor ligand interactions are very high, e.g. LH and hCG are using the same receptor, the LHIhCG receptor. For these reasons the colTesponding receptors are regarded to have co-evolved also from a common ancestor.

The low abundance of the glycoprotein hormone recep-

tors prevented a purification and determination of the primary structure until now. In the last years, molecular biology helped to establish the amino acid sequences of the LH/hCG receptor, the FSH, and the TSH receptors from the respective cDNAs (McFarland et al., 1989; Loosfelt et al., 1989; Sprengel et al., 1990; Minegish et al., 1991; Parmentier et al., 1989; Nagayama et al., 1989; Misrashi et al., 1990; Frazier et al., 1990; Nagayama and Rapoport, 1992). It turned out that the glycopro-

tein hormone receptors belong to a superfamily of Gprotein coupled transmembrane receptor proteins showing structural analogies to rhodopsin as well as to the (3-

adrenergic receptors: The members of these receptor family are transmembrane proteins composed of one single polypeptide chain. The extracellular amino-terminal domain contains the ligand binding site followed by a cluster of seven hydrophobic regions which are proposed

to form membrane spanning helices and the carboxy-

Downloaded by: University of Pittsburgh. Copyrighted material.

Institute of Biochemistry II, University of Heidelberg/Germany

W. E. Merz, Glycoprotein Hormone Receptors

5

ct-subunit with a truncated carboxy terminus and a native 13-subunit (des(88-92)-ct + 13-native) has completely lost the receptor binding ability probably due to an improper NH

Extracellular domain

conformation of the complex (Merz, 1979; Merz and Dörner, 1979).

The exact contact regions of the receptors and the

Transmembrane domain

Cytoplasmic domain

Fig. 1 Model of glycoprotein hormone receptors. Schematic view of the membrane insertion and the coupling to the signal-

ling complex. The receptors consist of a single polypeptide chain with a large extracellular amino terminal domain. The

peptide loop comprising the residues 38-57 of hCG-13 (disulfide bridge between Cys38 and Cys57), the "Keutmann loop". This peptide bound to antibodies against hCG and 13-subunit, formed an amphipathic helix in lipophilic environment and stimulated testosterone production with a half-maximal response of 3.55 X l0 M (Keutmann et al., 1986). This peptide region is considered to be a component of the receptor binding domains of hCG and hLH.

The extracellular domain of the LH/hCG receptor

ween helices 2+3, 4+5, 6+7) and cytoplasmic connecting

probably covers most of the surface of the hormone, since except of two ((3-3 and (3-5) out of 14 epitopes exposed at the surface of hCG are inaccessible to monoclonal anti-

loops

bodies when the hormone is in the receptor-bound state

transmembrane domain is made up of 7 transmembrane helices

with extracellular connecting loops E-I, E-II, and E-III (betC-I, C-I, and C-III (between helices 1 + 2, 3 + 4, 5 + 6). The carboxy terminus extends into the cytoplasm (cytoplasmic domain). The connecting loop C-III was proposed to achieve the coupling of the receptors to a stimulatory G-protein (GP) of the signalling complex (AC, adenylyl cyclase)

terminal domain which extents into the cytosol (Fig. 1). The extracellular domain has some putative sites for N-

glucosylation, the role of the carbohydrate moiety is discussed below. The receptors are coupled to the signal-

ling complex consisting of a stimulatory GTP-binding protein (Gs-Protein) and an adenylyl cyclase and, less well established, to phospholipase C, respectively. The coupling was suggested to occur via an interaction of the large third cytoplasmic connecting ioop, C-III (see Fig. 1), with the G-protein.

(Schwarz et al., l991a, b). This favors the possibility that multiple contact sites between the receptor and the hormone seems to exist. The extracellular domain is encoded by 10 of the 11 exons of the LH/hCG receptor gene. In that domain, 14 tandem-like oriented leucinerich (LR) motifs are present, structures which are used to be highly conserved in the superfamily of leucine-rich glycoproteins. The LR motifs are thought to be involved in amphipathic structures which allow intense proteinprotein interactions. By expression of truncated LHIhCG receptor molecules comprising segments of the extracellular domain of different length, Braun et aI. (1991) could show that the LR repeats 1-6 of the LH/hCG receptor confer hCG responses whereas the repeats 8-14 are not involved in high affinity binding of hCG. Expression of chimeric receptors in which the LR repeats 1-8 of the

LH/hCG receptor were replaced by the homologous Receptor-Hormone Interactions The glycoprotein hormones are bound by the extracellular domains of the receptors. Pierce et al. (1971) have shown

by means of hybrid molecules recombined e.g. from and LH-(3 that the receptor specificity residues in the (3-subunit portions of the glycoprotein hormones. A mandatory prerequisite for receptor binding is the intact TSH-

quarternary structure of the hormones. In other words, the correct association of the subunits is required for receptor binding. Recombination of hCG (or LH) subunits seems to proceed in a two-step process (Bewley et al., 1974; Ingham et aI., 1976) which consists of a rapid physical association of the subunits followed by a slow process which is necessary to fully restore the conformational, immunological and biological properties of hCG (Merz 1984). A recombination product which contains an

sequences of the FSM receptor gave raise to functional receptors which bound hCG and not FSH. A chimeric LH/hCG receptor containing the LR repeats 1-11 of the

FSH receptor bound FSH and not hCG. A detailed analysis of these experiments showed that amino acids around repeat 6 are crucial for the FSH specificity. A similar analysis of the extracellular domain of the TSH receptor confined the binding region of TSH to the LR repeats 7-10 and the region involved into signal trans-

duction to the LR repeats 10-14 (Nagayama et al., 1991). In the past, a number of groups have used bifunc-

tional reagents or photoaffinity labelling to cross-link ligand and receptor and label contact sites (reviewed by Ascoli and Segaloff, 1989). These experiments gave rise to a variety of receptor models based on receptor dimers which were detected in the absence of reducing agents, having a molecular weight (Mr) of 305 kDA and to the

Downloaded by: University of Pittsburgh. Copyrighted material.

ligands have to be determined in detail. Most information is available about the interaction of LH and hCG with the LH/hCG receptor. Of special interest is an intercysteine

6

Exp. Clin. Endocrinol. loo (1992) 1/2

truncated receptors formed during biosynthesis of the receptor (see below). Biosynthetic labelling of the LH/ hCG receptor yielded a protein with a M of 90-92 kDa.

LH and hCG show intrinsic thyrotropic activity (Taliadouros et al., 1978) which was proposed to be responsible for hyperthyroidism in normal pregnancy, in women with trophoblastic tumors and men with testicular cancer. Acid variants of hCG (most probably sialylated to a higher extent), which could be isolated from patients with trophoblastic diseases, showed thyrotropic activity

(Mann et al., 1986). Desialylation of hCG induced an increase in the receptor binding especially to the TSH receptor (Amir et al., 1987). Partially desialylated and asialoagalacto hCG molecules were identified as potent antagonists of bTSH on thyroid functions in vitro and in an in vivo model, suppressing also HLA-DR expression (Hoermann et al., 1988; Hoermann et al., 1991).

The Role of the Carbohydrate Moiety for Coupling of the Receptor-Hormone and the Signalling Complex

Binding of the hormone to the receptor is thought to change the conformation of the receptor which enables the cytosolic domains to couple to the signalling complex. In this connection, the role of the carbohydrate parts of the receptor and the glycoprotein hormone ligand is of special interest. The rat ovarian LH/hCG receptor seems to carry 2 N-linked carbohydrate chains out of a total number of 6 putative glycosylation sites on the LH/hCG receptor (McFarland et al., 1989). They are located on the outward face of the receptor-hormone complex and seem not to interfere with ligand binding (Petäjä-Repo et al., 1991). The carbohydrate part of the glycoprotein hormones has a profound effect on the biological properties of the glycoprotein hormones at the level of the receptor and post-receptor signalling: The deglycosylated hormones

ligand, dghCG, into an agonist. The extracellular domain of the LHIhCG receptor contains a region of 10 residues which are present in soybean lectin (McFarland, 1989). This region was proposed to be involved into recognition of the carbohydrate part of hCG. The steric orientation of the carbohydrate part of hCG in its receptor-bound state is still unclear. Detailed analyses of the binding of native hCG and dghCG to the LHI

hCG receptor by means of monoclonal antibodies (Schwarz et al., 199lac; Schwarz et al., 1992) suggested a direct interaction of the glycan chain of hCG with the extracellular connecting loops yielding in a certain repulsion due to negative charges in the extracel-

lular connecting loop E-II and the sialic acids of the carbohydrate moiety. The dghCG seems to be buried in the extracellular domain to a larger extent than native hCG, since the (3-subunit epitopes 3 and 5 are inaccessible for the monoclonal antibodies against these sites. On the other hand, Petäjä et al. (1991) reported that the sialic acid residues of receptor-bound hCG may be cleaved by neuraminidase which requires a superficial orientation of the carbohydrate residues.

Outlook Tremendous progress has been made in the last years in the receptor field by the molecular biology approach. Nevertheless, a lot of questions are still open, not only concerning the mechanism of receptor-hormone interaction and the coupling of the receptor-hormone complex to the adenylyl cyclase or possibly to the phospholipase C. The receptor metabolism is extremely important for the biology of the cell. The regulation of de novo synthesis of

receptors and the role of truncated or "intracellular" glycoprotein hormone receptors and their putative involvement in regulating the expression of ligands and receptors remain still to be elucidated.

References

seem to bind to the receptors but fail to activate the

Amir, S.; Kubota, K.; Tramontano, D.; Ingbar, S. H.; Keutmann, H. T.: The carbohydrate moiety of bovine thyrotropin is essential for full bioactivity but not for

signalling complex at least in some experimental systems

receptor recognition. Endocrinol. 120 (1987) 345-352

(Sairam and Schiller, 1979; Chen, H.-C., et al., 1982; Thotakura and Bahi, 1982; Constant and Weintraub, 1986; Amir et al., 1987; Merz, 1988). The mechanism

Amir, S. M.; Kasagi, K.; Ingbar, S. H.: The role of

which causes the uncoupling of the receptor-hormone and

Ascoli, M.; Segaloff, D. L.: On the structure of the

the signalling complexes by binding of deglyco-(dg-)

subunit sialic acid in the thyrotropic and gonadotropic acivities of human chorionic gonadotropin. Endocrinol. 121 (1987) 160-165 luteinizing hormone/chorionic gonadotropin receptor. En-

hormones is not known. There is evidence that the

docrine Rev. 10 (1989) 27-44

conformation of dghCG differs from native hCG (Merz, 1988). Treatment of the LH/hCG receptor-dghCG complex with Fab fragments of polyclonal anti-hCG (Rebois

Braun, T.; Schofield, P. R.; Sprengel, R.: Amino-terminal leucine-rich repeats in gonadotropin receptors determine hormone selectivity. EMBO J. 10 (1991) 1885-1890

and Fishman, 1984) or with monoclonal antibodies

K. J.: Characterization and biological properties of chemi-

against an epitope located in the region hCG-J3 82-105 (Loenen et al., in preparation) converted the antagonistic

Chen, H.-C.; Shimohigashi, Y.; Dufau, M. L.; Catt, cally deglycosylated human chorionic gonadotropin. J. Biol. Chem. 257 (1982) 14446-14452

Downloaded by: University of Pittsburgh. Copyrighted material.

suggestion of a subunit structure of the LH/hCG receptor with M1. between 38 kDa and 55 kDa. Some of the "subunits" may represent proteolytic cleavage products raised during the experimental procedures, others are possibly

W. E. Merz, Glycoprotein Hormone Receptors

(TSH) derived from euthyroid and hypothyroid rats: effects of chemical deglycosylation of pituitary TSH. Endocrinol. 6 (1986) 2720-2727 Frazier, A. L.; Robbins, L. S.; Stork, P. J.; Sprengel, R.; Segaloff, D. L.; Cone, R. D.: Isolation of TSH and LH/ CG receptor cDNAs from human thyroid: Regulation by tissue specific splicing. Mol.

Endocrinol. 4 (1990)

1264-1276 I-Ioermann, R.; Amir, S. M.; lngbar, S. H.: Evidence that partially desialylated variants of human chorionic gonadotropin (hCG) are the factors in crude hCG that inhibit the

response to thyrotropin in human thyroid membranes. Endocrinol. 123 (1988) 1535 1543 Hoermann, R.; Amir, S. M.; Nomura, T.; Ingbar, S. H.: Design of a long/lived thyrotropin antagonist from derivateves of human chorionic gonadotropin. Endocrinol. 124 (1989) 223-232 Hoermann, R.; Schumann-Draeger, P. M.; Rehbach, K.; Mann, K.: Asialoagalacto-human chorionic gonadotropin,

a carbohydrate-modified variant of human chorionic gonadotropin, antagonizes the stimulatory actions of bovine thyroid-stimulating hormone on thyroid function and HLA-DR expression in human thyroid in vitro and in vivo. J. Clin. Invest. 88 (1991) 1947 1954 Keutmann, H. T.; Charlesworth, M. Ch.; Mason, K. A.; Ostrea, T.; Johnson, L.; Ryan, R. J.: A receptor-binding region in human choriogonadotropine/lutropin 3 subunit. Proc. Natl. Acad. Sci. 84 (1987) 2038-2042 Loenen, H. J.; Bidard, J. M.; Rommerts, F. F. G.: Do the oligosaccharides of hCG influence the conformation of the hormone? In preparation. Loosfelt, H.; Misrahi, M.; Atger, M.; Salesse, R.; HaiLuu Thi, M. T. V.; Jolivet, A.; Guichon-Mantel, A.; Sar, S.; Jallai, B.; Gamier, J.; McFarland, K. C.; Sprengel, R.; Phillips, H. S.; Köhler, M.; Rosemblit, N.; Nicolics, K.; Segaloff, D. L.; Seeburg, P.: Lutropin-choriogonadotropin receptor: An unusual member of the G-protein-coupled receptor family. Science 245 (1989) 494-499

Mann, K.; Schneider, N.; Hoermann, R.: Thyrotropic activity of isoelectric variants of human chorionic gonadotropin from trophoblastic tumors. Endocrino!. 118

(1986) 1558 1566 Merz, W. E.; Dörner, M.: Studies of the specific role of the subunits of choriogonadotropin for biological, immunological and physical properties of the hormone. Digestion of choriogonadotropin and its isolated subunits with serine carboxypeptidase. Hoppe/Seyl. Z. Physiol. Chem. 360 (1979) 1783-1797 Merz, W. E.: Studies of the specific role of the subunits of choriogonadotropin for biological immunological and physical properties of the hormone. Digestion of the n-subunit with carboxypeptidase A. Eur. J. Biochem. 101 (1979) 54 1-553 Merz, W. E.: Evidence for impaired subunit interaction in chemically deglycosylated human choriogonadotropin. Biol. Biophys. Res. Comm. 156 (1988) 1271-1278 Merz, W. E.: Structure-function relationships of gonadotropins: Human choriogonadotropin as a model. In: Secretion and action of gonadotropins. Physiology and Clinic. Eds. Runnebaum, B.; Rabe, T.; Kiesel, L.; Men, W. E. Berlin, Heidelberg, New York, Tokyo: Springer-Verlag 1984, pp. 59-82 Milgrom, E.: Cloning and Sequencing of porcine LH-hCG

receptor cDNA: Variants lacking transmembrane domain. Science 245 (1989) 525-528 Minegish, T.; Nakamura, K.; Takakura, Y.: Cloning and

sequencing of human FSH receptor cDNA. Biochem. Biophys. Res. Commun. 175 (1991) 1125-1130 Nagayama, Y.; Wadsworth, H. L.; Chazenbalk, G. D.; Russo, D.; Seto., P.; Rapoport, B.: Thyrotropin-luteinizing hormone/chorionic gonadotropin receptor extracellular

domain chimeras as probes for TSH receptor function. Proc. NatI. Acad. Sei. USA 88 (1991) 902-905 Nagayama, Y.; Rapoport, B.: The thyrotropin receptor 25 years after its discovery: new insight after its molecular cloning. Mol. Endocrinol. 6 (1992) 145-156

Parmentier, M.; Libert, F.; Maenhaut, C.; Lefort, A.; Gerard, C.; Perret, 1.; van Sande, I.; Dumont I. E.; Vassart, G.: Molecular cloning of the thyrotropin receptor.

Science 246 (1989) 1620-1622 Petäjä, Repo, U.; Merz, W.-E.; Rajaniemi, H.: Significance of the glycan moiety of the rat ovarian luteinizing hormone/chorionic gonadotropin (CG) receptor and human CG for receptor-hormone interaction. Endocrinol. 128 (1991) 1209-1217 Pekonen, F.; Alfthan, 1-l.; Stenman, U. -H.; Ylikorkala, O.: Human chorionic gonadotropin (hCG) and thyroid function in early human pregnancy: circadian variation and evidence for intrinsic thyrotropic activity of hCG. J. Clin. Endocrino!. Metab. 66 (1988) 853-856 Pierce, J. G.; Bahl, O. P.; Cornell, J. S.; Swaminathan, N.: Biologically active hormons prepared by recombination of the n chain of human chorionic gonadotropin and the hormon-specific chain of bovine thyrotropin or of bovine luteinizing hormone. J. Biol. Chem. 246 (1971) 2321-2324 Rebois, V.; Fishman, P. H.: Antibodies against human chorionic gonadotropin convert the deglycosylated hormone from an antagonist to an agonist. J. Bio!. Chem. 259 (1984) 8087-8090 Russo, D.; Chazenbalk, D.; Nagayama, Y.; Wadsworth, H. L.; Seto, P.; Rapoport, B.: A new structural model for the thyrotropin (TSH) receptor, as determined by covalent cross-linking of TSH to the recombinant receptor in intact cells: Evidence for a single polypeptide chain. Mol. Endocninol. 5 (1991) 1607 1612 Sairam, M. R.; Schiller, P. W.: Receptor binding, biological and immunological properties of chemically deglycosylated pituitary lutropin. Arch. Biochem. Biophys. 197 (1979) 294-301 Schwarz, S.; Krude, H.; Nelboeck, E.; Berger, P.; Merz, W. E.; Wick, G.: Relationship of orientation with affinity and activity of receptor-bound glycosylation variants of human chorionic gonadotropin (hCG), as visualized by monoclonal antibodies (MCA). J. Rec. Res. 11 (1991a)

437-458 Schwarz, S.; Krude, H.; Wick, G.; Berger, P.: Twelve of fourteen surface epitopes of receptor-bound human chorionic gonadotropin (hCG) being antibody-inaccessible suggest an extensive involvement of the long extracellular domain of the hCG receptor. Mol. Cell. Endocrinol. 82

(l991b) 71-79 Schwarz, S.; Krude, H.; Klieber, R.; Dirnhofer, S.; Lot-

tersberger, C.; Men, W. E.; Wick, G.; Berger, P.: Number and topography of epitopes of human chorionic gonadotropin (hCG) are shared by desialylated and deg!ycosylated hCG. Mol. Cell. Endocrino!. 80 (1991 e) 33-40 Schwarz, S.; Lottersberger, C.; Krude, H.; Sprengel, R.;

Downloaded by: University of Pittsburgh. Copyrighted material.

Constant, R. B.; Weintraub, B. D.: Differences in the metabolic clearance of pituitary and serum thyrotropin

7

8

Exp. Clin. Endocrinol. 100 (1992) 1/2

agonistic and antagonistic forms of human chorionic gonadotropin using rat testis and hCG receptor-transfected 293 cells. In: Molecular and Cellular Biology of Reproduction. Eds. Bardin, C. W.; Fabbrini, A. New York: Raven Press, 1992, in press. Sprengel, R.; Braun, Th.; Nikolics, K.; Segaloff, D. L.;

human chorionic gonadotropin: deglycosylation uncouples hormone-receptor complex and adenylate cyclase system. Biochem. Biophys. Res. Comm. 108 (1982)

399-405 Zor, U.; Shentzer, P.; Azrad, A.; Sairam, M. R.; Amsterdam, A.: Deglycosylated luteinizing hormone (LH) prevents desensitization of cyclic adenosine monophosphate response by LH: dissociation between receptor un-

Seeburg, P. H.: The testicular receptor for follicle stimulating hormone: structure and functional expression

coupling and down regulation. Endocrinol. 114 (1984) 2143-2147

of cloned cDNA. Mol. Endocrinol. 4 (190) 525-530 Taliadouros, G. S.; Canfield, R. E.; Nisula, B. C.: Thyroid-stimulating activity of chorionic gonadotropin and luteinizing hormone. J. Clin. Endocrinol. Metabol. 47 (1978) 855-860 Thotakura, N. R.; Bahl, O. P.: Role of carbohydrate in

Author's address: Dr. Wolfgang E. Merz, Institut für Biochemie II, Universität Heidelberg, Im Neuenheimer Feld 328, W - 6900 Heidelberg, Germany

Downloaded by: University of Pittsburgh. Copyrighted material.

Merz, W. E.; Wick, G.; Berger, P.: Structure and function of the hCG receptor: Epitope mapping of receptor bound

Properties of glycoprotein hormone receptors and post-receptor mechanisms.

Exp. Clin. Endocrinol. loo (1992) 4-8 Experimental and Clinical Endocrinology © 1992 Johann Ambrosius Barth Properties of Glycoprotein Hormone Recep...
85KB Sizes 0 Downloads 0 Views