Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships.

Gonadotrophin-releasing hormone (GnRH) is the main neurohormone controlling gonadotrophin release in all vertebrates, and in teleost fish also of growth hormone and possibly of other adenohypophyseal hormones. Over 20 GnRHs have been identified in vertebrates and protochoordates and shown to bind cognate G-protein couple receptors (GnRHR). We have searched the puffer fish, Fugu rubripes, genome sequencing database, identified five GnRHR genes and proceeded to isolate the corresponding complementary DNAs in European sea bass, Dicentrachus labrax. Phylogenetic analysis clusters the European sea bass, puffer fish and all other vertebrate receptors into two main lineages corresponding to the mammalian type I and II receptors. The fish receptors could be subdivided in two GnRHR1 (A and B) and three GnRHR2 (A, B and C) subtypes. Amino acid sequence identity within receptor subtypes varies between 70 and 90% but only 50-55% among the two main lineages in fish. All European sea bass receptor mRNAs are expressed in the anterior and mid brain, and all but one are expressed in the pituitary gland. There is differential expression of the receptors in peripheral tissues related to reproduction (gonads), chemical senses (eye and olfactory epithelium) and osmoregulation (kidney and gill). This is the first report showing five GnRH receptors in a vertebrate species and the gene expression patterns support the concept that GnRH and GnRHRs play highly diverse functional roles in the regulation of cellular functions, besides the "classical" role of pituitary function regulation.

A novel mammalian receptor for the evolutionarily conserved type II GnRH.

Mammalian gonadotropin-releasing hormone (GnRH I: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) stimulates pituitary gonadotropin secretion, which in turn stimulates the gonads. Whereas a hypothalamic form of GnRH of variable structure (designated type I) had been shown to regulate reproduction through a cognate type I receptor, it has recently become evident that most vertebrates have one or two other forms of GnRH. One of these, designated type II GnRH (GnRH II: pGlu-His-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2), is conserved from fish to man and is widely distributed in the brain, suggesting important neuromodulatory functions such as regulating K+ channels and stimulating sexual arousal. We now report the cloning of a type II GnRH receptor from marmoset cDNA. The receptor has only 41% identity with the type I receptor and, unlike the type I receptor, has a carboxyl-terminal tail. The receptor is highly selective for GnRH II. As with the type I receptor, it couples to G(alpha)q/11 and also activates extracellular signal-regulated kinase (ERK1/2) but differs in activating p38 mitogen activated protein (MAP) kinase. The type II receptor is more widely distributed than the type I receptor and is expressed throughout the brain, including areas associated with sexual arousal, and in diverse non-neural and reproductive tissues, suggesting a variety of functions. Surprisingly, the type II receptor is expressed in the majority of gonadotropes. The presence of two GnRH receptors in gonadotropes, together with the differences in their signaling, suggests different roles in gonadotrope functioning.

Immunoidentification of gonadotropin releasing hormone receptor in human sperm, pituitary and cancer cells.

PROBLEM: To generate monoclonal antibodies specific to the receptor of gonadotropin releasing hormone (GnRH) for immunoidentification of the GnRH receptor (RGnRH) in human sperm, pituitary and cancer cells. METHODS: Four oligopeptides corresponding to RGnRH amino acid residues 1-29, 182-193, 195-206 and 293-306 in the extracellular domains were synthesized, conjugated to hemocyanin from Keyhole Limpet and used as immunogens to generate specific monoclonal antibodies. Enzyme-linked immunosorbent assay was used for initial screening of hybridomas. RESULTS: A total of 15 hybrid cell lines secreting RGnRH-specific monoclonal antibodies were initially established. By using some of these monoclonal antibodies as immunohistochemical probes, RGnRH was localized in the acrosomal region of human sperm, in the anterior pituitary tissue and in cancer cells of human ovarian and cervical origins. RGnRH was shown to have a molecular size of 45,000 +/- 5,000 kDa by Western blot assay. Expression of RGnRH mRNA in several human tissues and cancer cells was established by the reverse transcriptase-polymerase chain reaction method. CONCLUSION: RGnRH-specific monoclonal antibodies may be a valuable tool of identifying the presence of RGnRH in various normal and malignant human tissues.

Isolation and characterisation of the marmoset gonadotrophin releasing hormone receptor: Ser(140) of the DRS motif is substituted by Phe.

In order to facilitate the understanding of gonadotrophin-releasing hormone (GnRH) agonist and antagonist action in the primate animal model, the marmoset GnRH receptor (GnRH-R) was cloned and characterised. It was shown to have 95% and 85% sequence identity with the human and rat GnRH-Rs, respectively, and, when transiently expressed in COS-7 cells, it exhibited high-affinity des-Gly(10), [d-Trp(6)]-GnRH binding, with a K(d) value similar to those of both the rat and human forms, but with a greatly reduced B(max) value. The ED(50) for production of GnRH-induced total inositol phosphate (IP) for the marmoset GnRH-R was also similar to those of the rat and the human, but the maximal response compared with the rat receptor was markedly reduced. In all mammalian forms of the GnRH-R cloned to date, the conserved DRY region of G-protein-coupled receptors is substituted with DRS. The most interesting feature of the marmoset GnRH-R was the substitution of this motif with DRF. In order to investigate the DRS to DRF substitution, a Ser(140)Phe rat GnRH-R mutant was generated. The mutant had a K(d) value similar to that of the wild-type rat receptor, although the B(max) value was slightly lower, indicating that expression of functional mutant receptor at the cell surface was reduced. The ED(50) value for IP production was also similar to that of the wild-type receptor, with a reduction in maximal response. The level of internalisation for the rat wild-type and mutant GnRH-R constructs was also assessed and the Ser(140)Phe mutant was shown to have an increased rate of receptor internalisation, suggesting a role for this residue in regulating internalisation. These results show that the marmoset GnRH-R exhibits a substitution in the DRS motif and that this substitution may play a part in desensitisation and internalisation events.

The gene encoding the ovine gonadotropin-releasing hormone (GnRH) receptor: cloning and initial characterization.

We have isolated four lambda clones, which, in their aggregate, contain the entire coding sequence of the ovine gene encoding the gonadotropin-releasing hormone (GnRH) receptor (GnRHR). Like its human and murine counterparts, ovine GnRHR exists as a single-copy gene and is comprised of three exons and two introns. Furthermore, the locations of all exon-intron boundaries are perfectly conserved among the human, ovine and murine genes. The most striking difference among these genes is the location of the transcription start points (tsp) and, thus, the length of 5' untranslated region (UTR). This variation in size of the 5' UTR between the murine, human and ovine genes raises the possibility that different mechanisms have evolved for cell-specific expression of this gene. Isolation of the ovine GnRHR and its associated 5' flanking region is the essential first step in defining the molecular mechanisms underlying cell-specific and hormonal regulation of its expression in ruminants.

Development of methods for purification of membrane associated gonadotropin-releasing hormone binding proteins.

Gonadotropin-releasing hormone (GnRH) is the primary regulator of mammalian reproduction. It stimulates the release of luteinizing hormone and follicle stimulating hormone via receptors on the cell membranes of pituitary gonadotrope cells. This paper describes the development of a protocol for purification of GnRH binding proteins from sheep pituitary membranes. Membranes were best solubilized using a zwitterionic detergent. Solubilized membranes were applied to an affinity column prepared with a GnRH analogue. The most effective analogue was the agonist [D-Lys6,Pro9-NHEt]-GnRH. The column was washed with a gradient of sodium chloride up to 0.4 M and GnRH binding activity was eluted from the column using the acidic buffer. Eluted fractions bound labelled GnRH agonist after neutralization of the buffer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis revealed a major protein band with a relative molecular weight of 67 kD. Amino acid sequence analysis showed that the protein is different from the cloned GnRH receptor, but homologous with a similar protein recently purified from bovine pituitary. This protein may have a function which is modulated by binding of GnRH, GnRH fragments or GnRH-related peptides.

Purification and characterization of the bovine pituitary luteinizing hormone releasing hormone M(r) 60,000 binding protein.

Luteinizing hormone releasing hormone (LHRH) regulates the release of luteinizing hormone and follicle stimulating hormone from the pituitary. This process takes place through interaction with high affinity membrane receptors. In addition LHRH inhibits the growth of several cancer cell lines through the interaction with M(r) 60,000 LHRH receptors. Here we describe the purification to homogeneity of the M(r) 60,000 bovine pituitary LHRH binding protein in amounts allowing N-terminal sequencing and peptide mapping. The procedure describes solubilization of luteinizing hormone releasing hormone receptors from homogenized bovine pituitaries in an active form by using the detergent Triton X-114. The receptors were retained in the Triton X-114 phase during temperature-dependent phase separation. Preparative phase separations were performed directly on solubilized bovine pituitary extracts. SDS-PAGE of the purified LHRH receptor after LHRH-immobilized affinity chromatography showed the presence of a single band with M(r) 60,000. Partial sequencing of this band after trypsin digestion of gel pieces revealed unknown sequences with a possible homology to other receptors including some G-protein coupled receptors.

Characterization of the gonadotrophin-releasing hormone receptor in alpha T3-1 pituitary gonadotroph cells.

The present study has characterized the gonadotrophin-releasing hormone (GnRH) receptor in immortalized alpha T3-1 pituitary gonadotroph cells. GnRH and GnRH analogues produced both a dose- and time-dependent increase in total inositol phosphate (IP) accumulation. The rank order of potency of these analogues was the same as that obtained in parallel receptor-binding studies in alpha T3-1 cells. These responses were abolished following pretreatment with a GnRH antagonist. The use of a specific inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) assay demonstrated a rapid but short-lived rise in Ins(1,4,5)P3 production. Intracellular calcium ([Ca2+]i) was subsequently measured in alpha T3-1 cells using dual wavelength fluorescence microscopy combined with dynamic video imaging. GnRH produced a biphasic rise in [Ca2+]i. The initial calcium transient was complete within seconds while the smaller secondary plateau phase lasted several minutes. G-protein involvement in the IP response to GnRH in alpha T3-1 cells was investigated using sodium fluoride (NaF) and pertussis toxin (PTx) which activate and inactivate G-proteins respectively. Like GnRH, NaF produced a dose- and time-dependent increase in IP accumulation. Activation of phospholipase C in these cells by either GnRH or NaF was PTx-insensitive, suggesting that the G-protein involved was neither Gi nor Go but more probably Gq. Immunoblot analysis of alpha T3-1 cell membranes using antisera raised against the predicted C-terminal decapeptide of the alpha subunit of Gq demonstrated the presence of Gq in alpha T3-1 cells. Collectively these results show that the GnRH receptors expressed in alpha T3-1 cells are coupled to the phosphatidylinositol second messenger pathway via a specific G-protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of a gonadotropin-releasing hormone binding protein in goldfish serum.

A binding protein (BP) specific for gonadotropin-releasing hormone (GnRH) was previously demonstrated in goldfish serum. In the present study the binding protein was isolated and further characterized. The GnRH-BP, partially purified from goldfish serum using polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions, was concentrated in a single band, separate from all major components of serum proteins. The binding ability of the partially purified GnRH-BP was conserved; the isolated GnRH-BP specifically bound salmon GnRH and chicken GnRH-II, the native forms of GnRH present in goldfish, but not other forms of GnRH. The relative binding affinity of the partially purified GnRH-BP was [D-Arg6,Pro9-NEt]-salmon GnRH greater than chicken GnRH-II greater than or equal to salmon GnRH. The GnRH-BP, in raw serum or partially purified by PAGE, was specifically covalently labeled using 125I-[D-Lys6,Pro9-NEt]-salmon GnRH and the bifunctional cross-linking reagent, disuccinimidyl suberate, and then subjected to sodium dodecyl sulfate-PAGE under reducing conditions. The location of the radiolabeled GnRH-BP on PAGE gels was determined by cutting gels into sections and counting the radioactivity, or by autoradiography; the molecular weight of the GnRH-BP was estimated to be 40 KD. The covalently labeled GnRH-BP extracted from SDS-PAGE was subjected to high pressure liquid chromatography, and it coeluted with a single protein peak of the GnRH-BP partially purified by PAGE under nonreducing conditions. These studies demonstrate that the GnRH-BP is a minor component of serum proteins in goldfish; it is a single nonglycoprotein of about 40 kDa.

Purification of gonadotropin releasing hormone receptors using the avidin-biotin technique.

The avidin-biotin technique has been applied to the purification of gonadotropin releasing hormone (GnRH) receptors from other solubilized membrane proteins. The following steps were involved in this approach: (a) solubilization of rat pituitary GnRH receptor with the zwitterionic detergent CHAPS, 3-[(3-cholamidopropyl)-di-methylammonio]-1-propane sulfonate, (b) equilibration of the solubilized GnRH receptor with [biotinyl-D-Lys6]GnRH immobilized on avidin-agarose; and (c) elution of the receptors with high salt and GnRH analogues. Following two cycles of affinity chromatography the GnRH receptor was purified to homogeneity. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract and the calculated purification was approximately 10,000 to 15,000 fold. The development of a two step affinity chromatography for the purification of GnRH receptors can be used for detailed studies on the structure and function of the receptor. These studies will advance our understanding of the molecular basis of GnRH action.

Synthesis of gonadotropin-releasing hormone receptors by gonadotrope cell cultures: both preexisting receptors and those unmasked by protein kinase-C activators show a similar synthetic rate.

Responsiveness of gonadotropes to GnRH depends, in part, on the number of plasma membrane GnRH receptors. Since the steady state level of these plasma membrane receptors is a function of the rates of both receptor generation (synthesis, unmasking, and recycling) and loss (internalization, degradation, and inactivation) we have sought to quantify the rate of synthesis of GnRH receptors in pituitary cell cultures. Further, since the protein kinase-C activator phorbol 12-myristate 13-acetate (PMA) has been shown to unmask a class of GnRH receptors that appear to be uncoupled from phosphoinositide turnover, we have measured the rate of synthesis of this second receptor population. The present studies use the density shift technique; incorporation of densely labeled amino acids confers a higher density to newly synthesized proteins and allows their separation by physical means. Cultures of pituitary cells were prepared from female weanling rats. After cells had attached to the culture dishes, medium was replaced at 12-h intervals with medium containing either densely labeled or normal amino acids. After the incubation, GnRH receptors were covalently linked to a photoaffinity receptor agonist [( 125I]Tyr5-[azido-benzoyl-D-Lys6-GnRH]), then solubilized with 1+ sodium dodecyl sulfate. In some cultures PMA (50 nM) was included during the photoaffinity agonist-binding step. Newly synthesized (dense) receptors were separated from previously synthesized receptors by velocity sedimentation (0-20% sucrose in 1% sodium dodecyl sulfate-10 mM Tris-HCl, pH 7.0; centrifuged at 156,000 x g for 24 h). Gradients were fractionated, and the radioactivity contained in each fraction was quantified. Newly synthesized GnRH receptors exhibited a higher density, as evidenced by further migration into the gradient, than did normal GnRH receptors. There was a delay of approximately 6 h between exposure to dense amino acids and the appearance of densely labeled GnRH receptors at the plasma membrane. Equilibrium for incorporation of dense amino acids into GnRH receptors was 48 h of exposure to dense amino acids. The time required for synthesis of half the entire population of GnRH receptors was 28 +/- 2 h (mean +/- SEM; n = 4). Scatchard analysis and the pattern of GnRH-stimulated LH release from densely labeled cells indicated that they bound the photoaffinity label (Kd = 0.4 nM; approximately 1 fmol receptor/microgram DNA) and secreted gonadotropin normally. Additionally, treatment with PMA caused a significant increase (181 +/- 24%) in photoaffinity agonist binding, consistent with previous observations.

Photoaffinity labelling of gonadotropin releasing hormone binding sites in human epithelial ovarian carcinomata.

A photoaffinity labelled derivative of [D-Lys6]-GnRH was prepared with a bifunctional photolabile reagent (4-azidobenzoyl)-N-hydroxysuccinimide. In rat pituitary membranes, this analog retained high binding affinity (Ka = 0.12 x 10(9) M-1) consistent with a single class of receptors. The analog was iodinated and used for the identification of GnRH binding sites in human epithelial ovarian carcinomata. By sodium dodecyl sulfate electrophoresis in 10% polyacrylamide gel the presence of two labelled components could be demonstrated: a high molecular weight component of 63,200 and a smaller component of 46,000. Competition experiments with unlabelled ligand suggest that it is the high molecular weight component which specifically binds GnRH.

Solubilization and characterization of the rat pituitary gonadotrophin-releasing hormone receptor.

Specific receptors for gonadotrophin-releasing hormone (GnRH) were extracted from the rat pituitary gland with several detergents and characterized by binding studies with the potent GnRH antagonist [Ac-D-pCl-Phe1.2, D-Trp3, D-Lys6, D-Ala10]-GnRH (GnRHant). The particulate GnRH receptors were most effectively solubilized with 5 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS), which extracted 63% of the original membrane binding activity when assayed with 125I-labelled GnRHant. The binding affinities of particulate and CHAPS-solubilized receptors analysed with 125I-labelled GnRHant were 1.5 +/- 0.4 x 10(9) M-1 and 1.2 +/- 0.2 x 10(9) M-1 respectively. Gel filtration of the CHAPS-solubilized receptor revealed a major peak of specific binding activity with Mr of about 700,000. A hormone-receptor complex of similar Mr was observed when CHAPS-solubilized receptors were labelled with photoreactive radioiodinated [D-Lys6]-des-Gly10-GnRH-N-ethyl-amide and then analysed by gel chromatography. However, when pituitary particles were photolabelled and solubilized in 2% Triton X-100 before analysis on Sephacryl S-300, the Mr of the receptor was approximately 250,000, similar to the value obtained by sucrose density gradient centrifugation of the CHAPS-solubilized receptor. After solubilization in sodium dodecyl sulphate (SDS) the photolabelled receptor was eluted from Sephacryl S-300 as a 60 kDa peak which on SDS-gel electrophoresis contained a 52 kDa component, corresponding to the major binding subunit extracted directly from photolabelled pituitary membranes. The difference in higher molecular weight forms observed under non-denaturing and denaturing conditions could reflect the need for additional membrane components to maintain the active conformation of the GnRH receptor site. Whereas the minimum Mr of the solubilized receptor is about 250,000 under non-denaturing conditions, analysis of the photolabelled GnRH-receptor complex by SDS chromatography and electrophoresis indicates that a binding subunit with Mr of 50,000-60,000 is present in the GnRH holoreceptor.

Photoaffinity labeling of pituitary GnRH receptors: significance of the position of photolabel on the ligand.

Photoreactive derivatives of GnRH and its analogues were prepared by incorporation of the 2-nitro-4(5)-azidophenylsulfenyl [2,4(5)-NAPS] group into amino acid residues at positions 1, 3, 6, or 8 of the decapeptide sequence. The modification of Trp3 by the 2,4-NAPS group led to a complete loss of the luteinizing hormone (LH) releasing as well as LH-release-inhibiting activity of the peptide. The [D-Lys(2,4-NAPS)]6 analogue was a very potent agonist that, after covalent attachment by photoaffinity labeling, caused prolonged LH secretion at a submaximal rate. [Orn(2,4-NAPS)]8-GnRH, a full agonist with a relative potency of 7% of GnRH, after photoaffinity labeling caused prolonged maximal LH release from cultured pituitary cells. In contrast, [Orn(2,5-NAPS)]8-GnRH, although being equipotent with the 2,4-NAPS isomer in terms of LH releasing ability, was unable to cause prolonged LH release after photoaffinity labeling. Thus, [Orn(2,4-NAPS)]8-GnRH is a very effective photolabeling ligand of the functionally significant pituitary GnRH receptor. Based on this compound, a pituitary peptidase resistant derivative, D-Phe6,[Orn(2,4-NAPS)]8-GnRH-(1-9)-ethylamide, was synthesized. This derivative showed high-affinity binding to pituitary membranes with a Kd comparable to those of other GnRH analogues. A radioiodinated form of this peptide was used for pituitary GnRH-receptor labeling. This derivative labeled 59- and 57-kDa proteins in rat and 58- and 56-kDa proteins in bovine pituitary membrane preparations, respectively. This peptide also labeled pituitary GnRH receptors in the solubilized state and therefore appears to be a suitable ligand for the isolation and further characterization of the receptor.

Human placental LHRH receptor: agonist and antagonist labeling produces differences in the size of the non-denatured, solubilized receptor.

Membranes of human term placenta were labeled with several photosensitive and non-photosensitive analogues of LHRH. In both groups agonistic and antagonistic peptide structures were tested. The photolabeling amino acid azidophenylalanine was placed either in positions 2, 6 or 7. All compounds were specifically displacing iodinated Buserelin from human placental membranes and from rat pituitary membranes. The iodinated photolabels were displaced by cold Buserelin, some compounds however had a high non-specific binding. Photolabeling experiments on human placental membranes with radioactive photolabels produced all a band at 58,000 daltons in SDS-gel electrophoresis. Solubilization with a non-denaturing detergent and gel filtration produced a major radioactivity peak which was attributed to the LHRH receptor. All labeling experiments with agonist labels produced Kav's indifferent from each other but significantly different from the Kav's of antagonist labeled membranes. This result was confirmed with similar experiments carried out with radioactive Buserelin and a radioactive antagonist under non-photolytic conditions. It is therefore concluded that the placental LHRH receptor contains an LHRH binding component of 58,000 daltons but that the native receptor is composed of several proteins. It is also concluded that agonist occupation of the receptor induces the association of a further component which might be involved in the transmembrane signalling system. The agonist labeled, solubilized native LHRH receptor has a Stokes radius of 52 A and the same, antagonist labeled receptor a radius of 48 A.

Production and characterization of antibodies to gonadotropin-releasing hormone receptor.

Antibodies to the gonadotropin-releasing hormone (GnRH) receptor of bovine pituitary membranes have been raised in rabbits by immunization with affinity-purified receptor preparations. These antibodies did not compete with 125I-labeled GnRH analog (Buserelin) for binding to the receptors but did precipitate rat and bovine solubilized receptors labeled with 125I-Buserelin. Binding of the antibodies to the receptors was also demonstrated by immunoprecipitation of 125I-labeled purified receptors and photoaffinity-labeled receptors. The antibodies did not have a GnRH-like activity but rather inhibited, in a dose-dependent manner, GnRH-stimulated luteinizing hormone release from cultured rat pituitary cells. In addition, the antibodies did not inhibit luteinizing hormone release stimulated by high K+ concentration. This suggests that the antibodies recognize domains of the receptor other than the binding site of the hormone and thereby inhibit the biological response. These GnRH receptor antibodies provide a useful tool for studying GnRH receptor structure, function, localization, and biosynthesis.

Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor.

Gonadotropin-releasing hormone (GnRH) receptors were solubilized from rat pituitary membrane preparations in an active form by using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). The solubilized receptor exhibits high affinity, saturability, and specificity. The soluble supernatant retained 100% of the original binding activity when stored at 4 or -20 degrees C in the presence of 10% glycerol. The receptors were resolved into two components on the basis of chromatography on wheat germ agglutinin-agarose. Homogeneous receptor preparation was obtained by two cycles of affinity chromatography on immobilized avidin column coupled to [biotinyl-D-Lys6]GnRH. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract, and the calculated purification -fold was approximately 10,000 to 15,000. Analysis of iodinated purified GnRH receptors by autoradiography indicated the presence of two bands, Mr = 59,000 and 57,000. This was confirmed by photoaffinity labeling of the partially purified receptors and suggests that both components can specifically bind the hormone.