Effects of Asn87 and Asp318 mutations on ligand binding and signal transduction in the rat GnRH receptor.

Section: The Effect Of Transmembrane and Cytoplasmic Mutations On Fprcontrasting

confidence: 99%

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confidence: 99%

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confidence: 99%

See 1 more Smart Citation

Identification of Putative Sites of Interaction between the Human Formyl Peptide Receptor and G Protein

Miettinen¹,

Gripentrog²,

Mason³

et al. 1999

“…Receptor Binding Assay-Ligand binding assays were carried out on cell membranes from receptor expressing cells as described previously (17). Briefly, purified membranes were incubated for 120 min in assay buffer (40 mM Tris⅐HCl, 2 mM MgCl 2 , 0.1% BSA, pH 7.4) at 4°C with 125 Ϫ10 to 10 Ϫ6 M. The membranes were then filtered, and the radioactivity was counted.…”

Section: Methodsmentioning

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

133

113

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...

“…While the inter-related roles of these H2 and H7 side chains in receptor activation (9 -12) suggest that they constitute a structural and functional microdomain, this conclusion has been considered controversial (13,14). The initial study of this microdomain in the GnRH receptor reported that the presence of an Asp residue in both loci eliminated detectable binding.…”

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confidence: 99%

The Functional Microdomain in Transmembrane Helices 2 and 7 Regulates Expression, Activation, and Coupling Pathways of the Gonadotropin-releasing Hormone Receptor

Flanagan¹,

Zhou²,

Chi³

et al. 1999

Structural microdomains of G protein-coupled receptors (GPCRs) consist of spatially related side chains that mediate discrete functions. The conserved helix 2/helix 7 microdomain was identified because the gonadotropin-releasing hormone (GnRH) receptor appears to have interchanged the Asp 2.50 and Asn 7.49 residues which are conserved in transmembrane helices 2 and 7 of rhodopsin-like GPCRs. We now demonstrate that different side chains of this microdomain contribute specifically to receptor expression, heterotrimeric G protein-, and small G protein-mediated signaling. An Asn residue is required in position 2.50(87) for expression of the GnRH receptor at the cell surface, most likely through an interaction with the conserved Asn 1.50 (53) residue, which we also find is required for receptor expression. Most GPCRs require an Asp side chain at either the helix 2 or helix 7 locus of the microdomain for coupling to heterotrimeric G proteins, but the GnRH receptor has transferred the requirement for an acidic residue from helix 2 to 7. However, the presence of Asp at the helix 7 locus precludes small G protein-dependent coupling to phospholipase D. These results implicate specific components of the helix 2/helix 7 microdomain in receptor expression and in determining the ability of the receptor to adopt distinct activated conformations that are optimal for interaction with heterotrimeric and small G proteins.The gonadotropin-releasing hormone (GnRH) 1 receptor belongs to the rhodopsin-like family of G protein-coupled receptors (GPCR) (1). This family includes the light-sensitive opsins, protease-activated receptors, and receptors for neurotransmitters, peptides, and glycoproteins. High resolution structural data have not yet been obtained for any GPCR. However, projection maps of rhodopsin, amino acid sequence alignment, and computational modeling indicate that GPCRs have 7 membrane-spanning ␣-helices (2-6). There is a high degree of homology within the transmembrane helices and certain amino acids are highly conserved throughout the family (2, 3, 7). This diverse family shares the common function of propagating a signal across lipid membranes and the amino acid side chains which are conserved among the GPCRs are likely to constitute key structural motifs which subserve this universal GPCR function. Several models of GPCRs, including the GnRH receptor (4, 8), have been constructed as aids for investigating receptor structure-function relations. Molecular models of GPCRs can be used to integrate experimental observations and generate structural hypotheses. However, the complexity of these structures and the limited number of experimentally determined constraints can lead to inconsistent behavior of the models (4, 7). To overcome these limitations, we have pursued the approach of identifying discrete structural motifs within receptor models, which might constitute functional microdomains. The microdomains are characterized in detail and subsequently incorporated into whole receptor models. In the GnRH receptor, for exa...