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

Abstract

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...

Lack of a C-terminal Tail in the Mammalian Gonadotropin-releasing Hormone Receptor Confers Resistance to Agonist-dependent Phosphorylation and Rapid Desensitization

“…generation by agonist stimulation of transiently expressed receptors was determined as described previously (16). Briefly, cells were removed from 100-mm dishes 24 h following transfection and transferred to 24-well plates (1 ϫ 10 5 cells/well).…”

“…Materials -Reagents of analytical grade were obtained from suppliers listed previously (6,16,18,19) Derivation of Epitope-tagged Receptor Constructs-To examine receptor phosphorylation, we incorporated an epitope tag sequence into that of the receptor to allow immunoprecipitation with a commercially available antibody. A double-stranded oligonucleotide fragment corresponding to a triple repeat of an epitope of the amino acid sequence of the influenza hemagglutinin (HA) protein (YPYDVPDYA) was synthesized and ligated in frame, into the N terminus of the rGnRH-R, rGnRH-R/rTRH-R tail, and rGnRH-R/cfGnRH-R tail chimeric receptors in the vector pcDNA3.…”

“…It is of interest, therefore, that mammalian GnRH-Rs have no C-terminal tail and only a relatively short third intracellular loop with relatively few serine/ threonine residues as potential phosphorylation sites (14,15). We have recently reported that the lack of acute regulation of the GnRH-R is associated with the absence of a C-terminal tail (16) and the primary aim of the current study was to investigate whether the GnRH-R is resistant to agonist-dependent phosphorylation. In addition, we have explored the consequence of the lack of a C-terminal tail on both receptor phosphorylation and function by using the African catfish GnRH-R (cfGnRH-R) and the rat thyrotropin-releasing hormone receptor (rTRH-R) (both of which have C-terminal tails) and chimeras of the rGnRH-R having the C-terminal tail of either the cfGnRH-R or rTRH-R.…”

Three distinct types of GnRH receptor characterized in the bullfrog

“…First, bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3 contain C-terminal intracellular tails of 74, 57, and 79 amino acids, respectively. It is well established that this cytoplasmic domain, which is absent in the mammalian GnRHRs, is functionally important for agonist-dependent phosphorylation, desensitization, and internalization of other G protein-coupled receptors as well as nonmammalian GnRHRs (2,(25)(26)(27). Second, mammalian GnRHRs are characterized by a reciprocal exchange of Asp 87 in transmembrane domain II and Asn 318 in transmembrane domain VII, as compared with other G protein-coupled receptors (28,29).…”

Proper receptor signalling in a mutant catfish gonadotropin‐releasing hormone receptor lacking the highly conserved Asp90 residue

“…Ligand binding assays were carried out on cell membranes from HEK 293T cells expressing receptors as described [11]. Total inositol phosphate (IP) was extracted and separated as described by Millar et al [12].…”