Identification of N-glycosylation sites in the gonadotropin-releasing hormone receptor: role in receptor expression but not ligand binding

Davidson, James S.; Flanagan, Colleen A.; Zhou, Wei; Becker, I. I.; Elario, Ricardo; Emeran, Wedaad; Sealfon, Stuart C.; Millar, Robert P.

doi:10.1016/0303-7207(94)03449-4

Cited by 86 publications

(49 citation statements)

References 26 publications

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“…Similarly, gonadotropin-releasing hormone (GnRH) receptor mutants in which the consensus N-linked glycosylation sites were ablated showed unchanged ligand affinity and were able to stimulate downstream signaling pathways. However, the glycosylationdefective GnRH receptors displayed decreased B max values at 39-46% of wild-type depending on mutation site, implying decreased receptor expression and/or stability at the plasma membrane (40). Immunoblot shows the duration of SP-mediated activation of p38, p42/p44, and JNK in all cell lines.…”

Section: Discussionmentioning

confidence: 97%

Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

Tansky

Pothoulakis²,

Leeman³

2007

Proc. Natl. Acad. Sci. U.S.A.

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The neurokinin 1 receptor (NK1R), a G protein-coupled receptor involved in diverse functions including pain and inflammation, has two putative N-linked glycosylation sites, Asn-14 and Asn-18. We studied the role of N-linked glycosylation in the functioning of the NK1R by constructing three receptor mutants: two single mutants (Asn 3 Gln-14 and Asn 3 Gln-18) and a double mutant, lacking both glycosylation sites. Using a lentiviral transfection system, the mutants were stably transfected into NCM 460 cells, a nontransformed human colonic epithelial cell line. We observed that the magnitude of glycosylation as estimated by changes in gel migration depends on the number of glycosylation sites available, with the wild-type receptor containing the greatest amount of glycosylation. All mutant receptors were able to bind to substance P and neurokinin A ligand with similar affinities; however, the double mutant, nonglycosylated NK1R showed only half the B max of the wild-type NK1R. In terms of receptor function, the ablation of both N-linked glycosylation sites did not have a profound effect on the receptors' abilities to activate the MAP kinase families (p42/p44, JNK, and p38), but did affect SP-induced IL-8 secretion. All mutants were able to internalize, but the kinetics of internalization of the double mutant receptor was more rapid, when compared with wild-type NK1R. Therefore, glycosylation of NK1R may stabilize the receptor in the plasma membrane. These results contribute to the ongoing elucidation of the role of glycosylation in G proteincoupled receptors and the study of the neurokinin receptors in particular.G protein-coupled receptor ͉ substance P

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Section: Discussionmentioning

confidence: 97%

Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

Tansky

Pothoulakis²,

Leeman³

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Calnexin is a lectin-like protein that binds to newly glycosylated proteins in the ER; therefore, it is likely that the calnexin-GnRHR interaction involves one or more of the glycosylated residues in the receptor (Asn 18 and Asn 102 ; Davidson et al 1995). However, when the Asn 18 was mutated, the receptor was not expressed at the plasma membrane, and when the Asn 102 was mutated, that receptor appeared identical to the wildtype receptor.…”

Section: Discussionmentioning

confidence: 99%

Calnexin regulated gonadotropin-releasing hormone receptor plasma membrane expression

Conn

2006

Journal of Molecular Endocrinology

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A significant proportion of human gonadotropin-releasing hormone receptors (GnRHRs) are normally retained in the endoplasmic reticulum (ER); however, nearly all rat GnRHRs are routed to the plasma membrane. When mutations are introduced into either receptor, considerably more of the proteins are recognized by the quality control system (QCS) as misfolded and retained compared with wild-type (WT) receptor, resulting in decreased signaling in the presence of agonist. Calnexin, a component of the QCS, decreased plasma membrane expression of the GnRHRs, an effect that was mediated by a physical interaction between the receptor and the calnexin. Only the human receptor showed reduced signaling because it had fewer spare receptors compared with the rat GnRHR, allowing calnexin to affect signaling. Calnexin did not affect receptor signaling when K 191 was deleted from the human WT GnRHR. Removal of this amino acid decreases receptor misfolding and increases plasma membrane expression. K 191 is not present in the rat WT GnRHR. A pharmacological chaperone that corrects GnRHR misfolding, increased expression of the human WT GnRHR in the presence of calnexin. Calnexin apparently retains misfolded GnRHRs but routes correctly folded receptors to the plasma membrane. Mutation of a calnexin protein kinase C consensus phosphorylation site promoted increased retention of the human GnRHR, suggesting that calnexin phosphorylation controls the retention mechanism. We conclude that a proportion of the human and the rat WT GnRHR appears to be retained in the ER by calnexin, an effect that decreases GnRHR signaling capacity.

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“…An Asn residue (Asn 18 ) in the N-terminal domain of the human GnRH receptor, which has been shown to be glycosylated and important for receptor expression in mammalian GnRH receptors (24), is conserved in the GfA and GfB GnRH receptors (Asn 12 and Asn 23 , respectively) and the catfish GnRH receptor (Asn 23 ). Two additional glycosylation sites (Asn 11 and Asn 18 ) are conserved between the goldfish GfB receptor and the catfish GnRH receptors.…”

Section: Discussionmentioning

confidence: 99%

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

Illing

Troskie

Nahorniak

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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139

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Identification of N-glycosylation sites in the gonadotropin-releasing hormone receptor: role in receptor expression but not ligand binding

Cited by 86 publications

References 26 publications

Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

Calnexin regulated gonadotropin-releasing hormone receptor plasma membrane expression

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

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