Molecular cloning and characterisation of the rat pituitary gonadotropin-releasing hormone (GnRH) receptor

Eidne, Karin A.; Sellar, Robin; Couper, Gregory S.; Andersen, Linnea K.; Taylor, Philip L.

doi:10.1016/0303-7207(92)90116-n

Cited by 125 publications

(40 citation statements)

References 21 publications

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“…This phosphorylation event is rapid, occurring within the first few minutes of agonist addition. Of all the GPCRs cloned to date, the mammalian GnRH-R is the only receptor in which the functionally important intracellular cytoplasmic C-terminal tail is completely absent, as it is truncated at the cell membrane immediately after the seventh transmembrane domain (1). Studies by our group and by others (7,12,13) have shown that in contrast to other GPCRs, the GnRH-R does not show rapid desensitization of IP production.…”

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confidence: 87%

“…GnRH and its receptor (GnRH-R) are key mediators of the regulation of the reproductive axis controlling the synthesis and release of the gonadotropins, luteinizing hormone, and follicle-stimulating hormone. The GnRH-R is a member of the large family of G-protein-coupled receptors (GPCRs) (1) and is preferentially coupled to phosphoinositidase C via the G q /G 11 family of G-proteins. Following stimulation by GnRH, the production of inositol phosphates (IPs) and diacylglycerol is induced resulting in the elevation of cytosolic calcium and the activation of protein kinase C (2)(3)(4).…”

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confidence: 99%

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Gonadotropin-releasing Hormone Receptors with Intracellular Carboxyl-terminal Tails Undergo Acute Desensitization of Total Inositol Phosphate Production and Exhibit Accelerated Internalization Kinetics

Heding

Vrecl

Bogerd³

et al. 1998

Journal of Biological Chemistry

133

113

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The mammalian gonadotropin-releasing hormone receptor (GnRH-R) is the only G-protein-coupled receptor (GPCR) in which the intracellular C-terminal tail is completely absent. In contrast to other GPCRs, the GnRH-R does not show rapid desensitization of total inositol (IP) production, and the rates of internalization are exceptionally slow. We investigated whether the incorporation of a cytoplasmic tail into the C terminus of the GnRH-R affects desensitization events and receptor internalization rates. A GnRH-R/TRH-R chimera was created where the intracellular tail of the rat thyrotropin-releasing hormone receptor (TRH-R) was engineered into the C terminus of the rat GnRH-R. Three different rat GnRH-R cDNA stop codon mutations (one for each reading frame) were also made. The GnRHstimulated IP production of the wild-type rat GnRH-R expressed in either COS-7 or HEK 293 cells did not desensitize even after prolonged stimulation with GnRH. In contrast, the catfish GnRH-R (which does possess an intracellular tail) and the TRH-R rapidly (<10 min) desensitized following agonist stimulation. The GnRH-R/TRH-R chimera also desensitized following treatment with GnRH, resembling the pattern shown by the TRH-R and the catfish GnRH-R. Two of the stop codon mutants did not show desensitization of IP production, and the third mutant with the longest tail was not functional. Internalization experiments showed that the rat GnRH-R had the slowest endocytosis and recycling rates compared with the TRH-R, the catfish GnRH-R, and the chimeric GnRH/TRH-R. This study demonstrates that the addition of a functional intracellular C-terminal tail to the GnRH-R produces rapid desensitization of IP production and significantly increases internalization rates.Gonadotropin-releasing hormone (GnRH) 1 is a decapeptide released by the hypothalamus which acts on specific receptors in the anterior pituitary. GnRH and its receptor (GnRH-R) are key mediators of the regulation of the reproductive axis controlling the synthesis and release of the gonadotropins, luteinizing hormone, and follicle-stimulating hormone. The GnRH-R is a member of the large family of G-protein-coupled receptors (GPCRs) (1) and is preferentially coupled to phosphoinositidase C via the G q /G 11 family of G-proteins. Following stimulation by GnRH, the production of inositol phosphates (IPs) and diacylglycerol is induced resulting in the elevation of cytosolic calcium and the activation of protein kinase C (2-4). A number of GPCRs exhibit homologous desensitization upon prolonged stimulation with agonist. This involves uncoupling of G-proteins, rapid down-regulation of the receptor's effector systems such as total inositol phosphate (IP) production followed by receptor internalization, and presumably subsequent proteolytic degradation. In particular this has been demonstrated for the m3 muscarinic acetylcholine receptor (5) and the thyrotropin-releasing hormone receptor (TRH-R) (6 -9). The phosphorylation of specific amino acids in the cytoplasmic part of the C-terminal tail of s...

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mentioning

confidence: 87%

mentioning

confidence: 99%

Gonadotropin-releasing Hormone Receptors with Intracellular Carboxyl-terminal Tails Undergo Acute Desensitization of Total Inositol Phosphate Production and Exhibit Accelerated Internalization Kinetics

Heding

Vrecl

Bogerd³

et al. 1998

Journal of Biological Chemistry

133

113

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“…An interesting feature of the Type II GnRH receptor homolog is the presence of a carboxyl terminal tail which is absent in all of the mammalian Type I GnRH receptors (Eidne et al 1992, Kaiser et al 1992, Kakar et al 1992, Reinhart et al 1992, Tsutsumi et al 1992, Brooks et al 1993, Chi et al 1993, Illing et al 1993, Kakar et al 1993, Perrin et al 1993) but which is a universal feature in all other GPCRs and in non-mammalian GnRH receptors , Troskie et al 1997a. The carboxyl terminal tail has been shown to play a role in homologous and heterologous desensitization of many GPCRs and its absence in the Type I GnRH receptor may be associated with a lack of rapid desensitization of ligand-stimulated inositol phosphate production , McArdle et al 1995, Heding et al 1998.…”

Section: Gene Structure Cdna Sequence and Amino Acid Sequencementioning

confidence: 99%

“…The existence of several forms of the GnRH ligand within a single species suggested that there may also be more than one form of receptor. To date only one form of GnRH receptor (designated here as Type I) has been cloned from mammals (Eidne et al 1992, Kaiser et al 1992, Kakar et al 1992, Reinhart et al 1992, Tsutsumi et al 1992, Brooks et al 1993, Chi et al 1993, Illing et al 1993, Kakar et al 1993, Perrin et al 1993, Troskie et al 1998. We therefore designed a series of pairs of degenerate oligonucleotides to the mammalian GnRH receptor and cloned short sequences encoding the extracellular (EC) loop 3 domain (EC3) which suggested the presence of three different GnRH receptor genes in species of fish, amphibian, reptile and bird (Troskie et al 1998).…”

Section: Introductionmentioning

confidence: 99%

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

Millar

Conklin²,

Lofton–Day³

et al. 1999

Journal of Endocrinology

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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.

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“…The TRHR1 C335/Rluc was PCR-amplified from amino acids 1 to 334 of the rat TRHR1-coding region and cloned inframe into the pRluc vector (30). The rat gonadotropin-releasing hormone receptor (GnRHR) was PCR-amplified from a previously described clone (39) and inserted into pcDNA3. The GnRHR/EYFP construct has been previously described (30).…”

Section: G-protein-coupled Receptor (Gpcr)mentioning

confidence: 99%

Homo- and Hetero-oligomerization of Thyrotropin-releasing Hormone (TRH) Receptor Subtypes

Hanyaloglu¹,

Seeber²,

Kohout³

et al. 2002

Journal of Biological Chemistry

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G-protein-coupled receptors (GPCRs) are regulated by a complex network of mechanisms such as oligomerization and internalization. Using the GPCR subtypes for thyrotropin-releasing hormone (TRHR1 and TRHR2), the aim of this study was to determine if subtype-specific differences exist in the trafficking process. If so, we wished to determine the impact of homo-and hetero-oligomerization on TRHR subtype trafficking as a potential mechanism for the differential cellular responses induced by TRH. Expression of either ␤-arrestin 1 or 2 promoted TRHR1 internalization. In contrast, only ␤-arrestin 2 could enhance TRHR2 internalization. The preference for ␤-arrestin 2 by TRHR2 was supported by the impairment of TRHR2 trafficking in mouse embryonic fibroblasts (MEFs) from either a ␤-arrestin 2 knockout or a ␤-arrestin 1/2 knockout, while TRHR1 trafficking was only abolished in MEFs lacking both ␤-arrestins. The differential ␤-arrestin-dependence of TRHR2 was directly measured in live cells using bioluminescence resonance energy transfer (BRET). Both BRET and confocal microscopy were also used to demonstrate that TRHR subtypes form hetero-oligomers. In addition, these hetero-oligomers have altered internalization kinetics compared with the homo-oligomer. The formation of TRHR1/2 heteromeric complexes increased the interaction between TRHR2 and ␤-arrestin 1. This may be due to conformational differences between TRHR1/2 hetero-oligomers versus TRHR2 homo-oligomers as a mutant TRHR1 (TRHR1 C335Stop) that does not interact with ␤-arrestins, could also enhance TRHR2/␤-arrestin 1 interaction. This study demonstrates that TRHR subtypes are differentially regulated by the ␤-arrestins and also provides the first evidence that the interactions of TRHRs with ␤-arrestin may be altered by hetero-oligomer formation. G-protein-coupled receptor (GPCR)1 function is underpinned by the formation of protein-protein interactions and complexes that are dynamically regulated by a variety of stimuli. These molecular interactions are involved in receptor recognition, activation, and desensitization and are targets for the majority of current therapeutic compounds utilized to treat a variety of disorders. More recently, the escalating body of evidence for GPCR oligomerization also adds another level of complexity in understanding how GPCRs are activated and signal and traffick in the cell. GPCR hetero-oligomerization may explain the diverse physiological responses obtained from a single ligand. Indeed, a number of GPCR subtypes such as the somatostatin, opioid, angiotensin, and ␤-adrenergic receptors have been shown to form hetero-oligomers that result in a receptor unit with either altered ligand specificity, signaling, or internalization rates (1-5). GPCR activation is also regulated by receptorprotein interactions involved in desensitization and internalization. For the majority of GPCRs, the mechanism of internalization is via the ␤-arrestin and dynamin-dependent pathway where agonist-activated phosphorylated receptors interact with the ␤-arres...

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Molecular cloning and characterisation of the rat pituitary gonadotropin-releasing hormone (GnRH) receptor

Cited by 125 publications

References 21 publications

Gonadotropin-releasing Hormone Receptors with Intracellular Carboxyl-terminal Tails Undergo Acute Desensitization of Total Inositol Phosphate Production and Exhibit Accelerated Internalization Kinetics

Gonadotropin-releasing Hormone Receptors with Intracellular Carboxyl-terminal Tails Undergo Acute Desensitization of Total Inositol Phosphate Production and Exhibit Accelerated Internalization Kinetics

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

Homo- and Hetero-oligomerization of Thyrotropin-releasing Hormone (TRH) Receptor Subtypes

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