Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling.

Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.

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.

Gonadotropin-releasing hormone receptor in the teleost Haplochromis burtoni: structure, location, and function.

GnRH acts via GnRH receptors (GnRH-R) in the pituitary to cause the release of gonadotropins that regulate vertebrate reproduction. In the teleost fish, Haplochromis burtoni, reproduction is socially regulated through the hypothalamus-pituitary-gonadal axis, making the pituitary GnRH-R a likely site of action for this control. As a first step toward understanding the role of GnRH-R in the social control of reproduction, we cloned and sequenced candidate GnRH-R complementary DNAs from H. burtoni tissue. We isolated a complementary DNA that predicts a peptide encoding a G protein-coupled receptor that shows highest overall identity to other fish type I GnRH-R (goldfish IA and IB and African catfish). Functional testing of the expressed protein in vitro confirmed high affinity binding of multiple forms of GNRH: Localization of GnRH-R messenger RNA using RT-PCR revealed that it is widely distributed in the brain and retina as well as elsewhere in the body. Taken together, these data suggest that this H. burtoni GnRH receptor probably interacts in vivo with all three forms of GNRH:

Three distinct types of GnRH receptor characterized in the bullfrog.

It has been proposed recently that two types of GnRH receptors (GnRHR) exist in a particular species. Here we present data demonstrating that at least three types of GnRHR are expressed in a single diploid species, the bullfrog. Three different cDNAs, encoding distinct types of bullfrog GnRHR (bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3), were isolated from pituitary and hindbrain of the bullfrog. BfGnRHR-1 mRNA was expressed predominantly in pituitary, whereas bfGnRHR-2 and -3 mRNAs were expressed in brain. The bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3 proteins have an amino acid identity of approximately 30% to approximately 35% with mammalian GnRHRs and approximately 40% to approximately 50% with nonmammalian GnRHRs. Interestingly, bfGnRHR-2 has an 85% amino acid homology with Xenopus GnRHR. Less than 53% amino acid identity was observed among the three bfGnRHRs. All isolated cDNAs encode functional receptors because their transient expression in COS-7 cells resulted in a ligand-dependent increase in inositol phosphate production. Notably, all three receptors exhibited a differential ligand selectivity. For all receptors, cGnRH-II has a higher potency than mGnRH. In addition, salmon GnRH also has a strikingly high potency to stimulate all three receptors. In conclusion, we demonstrated the presence of three GnRHRs in the bullfrog. Their expression in pituitary and brain suggests that bfGnRHRs play an important role in the regulation of reproductive functions in the bullfrog.

Desensitization and internalization of human and xenopus gonadotropin-releasing hormone receptors expressed in alphaT4 pituitary cells using recombinant adenovirus.

Nonmammalian vertebrates express at least two forms of GnRH and distinct forms of GnRH receptor (GnRH-R) have coevolved with their ligands. Mammalian and nonmammalian GnRH-R have key structural differences (notably the lack of C-terminal tails in mammalian GnRH-R) and comparative studies are beginning to reveal their functional relevance. However, cellular context and receptor density influence G protein-coupled receptor function and may be important variables in such work using heterologous expression systems. Here we report a comparative study using alphaT4 cells (gonadotrope progenitors that lack endogenous GnRH-R) transfected with a mammalian (human) or nonmammalian (Xenopus laevis type I) GnRH-R. Because conventional transfection strategies proved inefficient, recombinant adenovirus expressing these receptors were constructed, enabling controlled and efficient GnRH-R expression. When expressed in alphaT4 cells at physiological density, these GnRH-Rs retain the pharmacology of their endogenous counterparts (as judged by ligand specificity in radioligand binding and inositol phosphate accumulation assays) but do not activate adenylyl cyclase and are not constitutively active. Moreover, the Xenopus GnRH-R rapidly desensitizes and internalizes in these cells, whereas the human GnRH-R does not, and the internalization rates are not dependent upon receptor number. These data extend studies in COS, HEK, and GH3 cells showing that other GnRH-R with C-terminal tails desensitize and internalize rapidly, whereas tail-less mammalian GnRH-R do not. Retention of these distinctions at physiological receptor density in gonadotrope lineage cells, supports the argument that the evolution of nondesensitizing mammalian GnRH-Rs is functionally relevant and related to the development of mammalian reproductive strategies.

Molecular cloning, distribution and pharmacological characterization of a novel gonadotropin-releasing hormone ([Trp8] GnRH) in frog brain.

To date nine structural variants of GnRH have been identified in vertebrates and two additional forms have been isolated from a tunicate. In amphibians only mammalian GnRH ([Arg8] GnRH) and type II GnRH (chicken GnRH II, [His5, Trp7, Tyr8] GnRH) have been identified. In the present study, a full-length cDNA encoding a novel type of GnRH was isolated from pituitary of Rana dybowskii. The GnRH gene encodes a GnRH peptide ([Trp8] GnRH) in which tryptophan is substituted for arginine of mammalian GnRH Northern blot analysis revealed the presence of a single 500 bp transcript for the [Trp8] GnRH precursor in forebrain but its absence in testis, ovary, kidney and liver. Restriction digests of genomic DNA demonstrated a single copy of the gene. The [Trp8] GnRH immunoreactive cells were identified in the preoptic area of the frog brain. Synthetic [Trp8] GnRH was tested for its ability to stimulate inositol phosphate production by COS-1 cells transfected with the cloned Xenopus pituitary GnRH receptor and the cloned human GnRH receptor. [Trp8] GnRH had a potency of about 60% compared with mammalian GnRH ([Arg8] GnRH) for the Xenopus receptor, whereas the potency of [Trp8] GnRH was approximately 5% compared with mammalian GnRH for the human receptor. Both mammalian GnRH and [Trp8] GnRH were 1000-fold less potent than type II GnRH for the Xenopus GnRH receptor. The similar potency of [Arg8] GnRH and the novel [Trp8] GnRH for the Xenopus pituitary receptor indicates that, unlike the human receptor, the Xenopus receptor does not discriminate between these amino acids in position eight thereby allowing substitution of the arginine in the mammalian GnRH.

Complementary deoxyribonucleic acid cloning, gene expression, and ligand selectivity of a novel gonadotropin-releasing hormone receptor expressed in the pituitary and midbrain of Xenopus laevis.

We have cloned the full-length complementary DNA (cDNA) for a GnRH receptor from Xenopus laevis pituitary cDNA and determined its gene structure. The cDNA encodes a 368-amino acid protein that has a 46% amino acid identity to the human GnRH receptor. The X laevis GnRH receptor has all of the amino acids identified in the mammalian GnRH receptors as sites of interaction with the GnRH ligand. However, this receptor cDNA shares the same distinguishing structural features of the GnRH receptor that have been characterized from other nonmammalian vertebrates. These include the pair of aspartate residues in the transmembrane domains II and VII compared with the aspartate/asparagine arrangement in mammalian receptors, the amino acid PEY motif in extracellular loop III (SEP in mammals), and the presence of a carboxyl-terminal tail. Previous studies have reported that mammalian GnRH was equipotent to other naturally occurring GnRH subtypes in stimulating LH release from the amphibian pituitary. However, in this study we show that the X. laevis GnRH receptor has ligand selectivity for the naturally occurring GnRHs similar to other nonmammalian GnRH receptors. The order of potency of the GnRHs in stimulating inositol phosphate production in COS-1 cells transiently transfected with the X. laevis GnRH receptor cDNA was chicken GnRH II>salmon GnRH>mammalian GnRH. Transcripts of this GnRH receptor are expressed in the pituitary and midbrain of X. laevis.

A novel human GnRH receptor homolog gene: abundant and wide tissue distribution of the antisense transcript.

Gonadotropin releasing hormone (GnRH) regulates the reproductive system through a specific G-protein-coupled receptor (GPCR) in pituitary gonadotropes. The existence of two (or more) forms of GnRH in most vertebrates suggested the existence of GnRH receptor subtypes (I and II). Using sequence information for extracellular loop 3 of a putative Type II GnRH receptor from a reptile species, we have looked for a Type II GnRH receptor gene in the human genome EST (expressed sequence tag) database. A homolog was identified which has 45% and 41% amino acid identity with exons 2 and 3 of the known human GnRH pituitary receptor (designated Type I) and much lower homology with all other GPCRs. A total of 27 contiguous ESTs was found and comprised a continuous sequence of 1642 nucleotides. The EST sequences were confirmed in the cloned human gene and in PCR products of cDNA from several tissues. All EST transcripts detected were in the antisense orientation with respect to the novel GnRH receptor sequence and were highly expressed in a wide range of human brain and peripheral tissues. PCR of cDNA from a wide range of tissues revealed that intronic sequence equivalent to intron 2 of the Type I GnRH receptor was retained. The failure to splice out putative intron sequences in transcripts which spanned exon-intron boundaries is expected in antisense transcripts, as candidate donor and acceptor sites were only present in the gene when transcribed in the orientation encoding the GnRH receptor homolog. No transcripts extended 5' to the sequence corresponding to intron 2 of the Type I GnRH as the antisense transcripts terminated in poly A due to the presence of a polyadenylation signal sequence in the putative intron 2 when transcribed in the antisense orientation. These findings suggest that a Type II GnRH receptor gene has arisen during vertebrate evolution and is also present in the human. However, the receptor may have become vestigial in the human, possibly due to the abundant and universal tissue transcription of the opposite DNA strand to produce antisense RNA.

Two gonadotropin-releasing hormone receptor subtypes with distinct ligand selectivity and differential distribution in brain and pituitary in the goldfish (Carassius auratus).

In the goldfish (Carassius auratus) the two endogenous forms of gonadotropin-releasing hormone (GnRH), namely chicken GnRH II ([His5, Trp7,Tyr8]GnRH) and salmon GnRH ([Trp7,Leu8]GnRH), stimulate the release of both gonadotropins and growth hormone from the pituitary. This control is thought to occur by means of the stimulation of distinct GnRH receptors. These receptors can be distinguished on the basis of differential gonadotropin and growth hormone releasing activities of naturally occurring GnRHs and GnRHs with variant amino acids in position 8. We have cloned the cDNAs of two GnRH receptors, GfA and GfB, from goldfish brain and pituitary. Although the receptors share 71% identity, there are marked differences in their ligand selectivity. Both receptors are expressed in the pituitary but are differentially expressed in the brain, ovary, and liver. Thus we have found and cloned two full-length cDNAs that appear to correspond to different forms of GnRH receptor, with distinct pharmacological characteristics and tissue distribution, in a single species.

Identification of three putative GnRH receptor subtypes in vertebrates.

The majority of vertebrates have two or three forms of gonadotropin-releasing hormone (GnRH), which appear to have arisen by successive gene duplication events. This suggests the possibility of concordant gene duplications of the GnRH receptor to produce two or more cognate receptors. Since the extracellular loop 3 (EC3) domain of mammalian GnRH receptors plays a role in distinguishing the different forms of GnRH, we have contemplated that the sequence of this domain will differ significantly in the putative cognate receptors. Degenerate oliognucleotides encoding the sequences of the transmembrane domains preceding and following EC3 were used for PCR amplification of genomic DNA from zebrafish (Brachydanio rerio), goldfish (Carassius auratus), African clawed frog (Xenopus laevis), chicken (Gallus domesticus), and lizard (Agama atra). Isolation and sequencing of specific clones revealed that they fell into three groups. Two of these were most similar to the mammalian pituitary GnRH receptor and were therefore designated Type IA and Type IB. The third form (designated Type II) was most different from the others and was identified in Xenopus, lizard, and human DNA. These findings support the concept of the existence of three distinct GnRH receptors, which have evolved in conjunction with three distinct GnRH ligand classes present in many vertebrates.

Chicken GnRH II-like peptides and a GnRH receptor selective for chicken GnRH II in amphibian sympathetic ganglia.

Amphibia, like most vertebrate species, have two forms of GnRH, namely [Arg8]GnRH (mammalian GnRH) and [His5,Trp7,Tyr8] GnRH (chicken GnRH II). The differential distribution of the two peptides in the amphibian brain suggests that they may play different roles. Mammalian GnRH, which is found predominantly in the hypothalamus, is most likely the prime regulator of gonadotropin release, while chicken GnRH II, which occurs predominantly in the midbrain and hindbrain, may play a neuromodulatory role. In amphibian sympathetic ganglia, GnRH has been demonstrated to be a neurotransmitter where its release from the presynaptic nerve terminals reversibly inhibits M current, a time- and voltage-dependent potassium current. The occurrence of GnRH in sympathetic ganglia extracts from two amphibian species was investigated. Chicken GnRH II-like immunoreactivity was detected in extracts of bullfrog (Rana catesbeiana) and platanna (Xenopus laevis) sympathetic ganglia after high performance liquid chromatography. Under the chromatographic conditions used, a second unknown peptide co-eluted with synthetic mammalian GnRH, but showed no cross-reactivity with specific mammalian GnRH antisera. To test the possibility of the presence of a chicken GnRH II receptor in sympathetic ganglion neurones, competition binding of membranes extracted from the sympathetic ganglia of the two amphibian species was investigated with 125I-labelled GnRH agonists. The binding of 125-I-[His5,D-Arg6,Trp7,Tyr8]GnRH (a chicken GnRH II agonist) to membranes from the sympathetic ganglia of both amphibian species was specific and had a higher affinity than chicken GnRH II, mammalian GnRH and a mammalian GnRH agonist [D-Ala6,NMe-Leu7,Pro9-NHEt]GnRH. These findings suggest that endogenous chicken GnRH II may play a role in synaptic transmission in the sympathetic ganglia via a receptor specific for chicken GnRH II.