Abstract
The growth hormone secretagogue receptor (GHSR) plays an important role in regulating food intake and energy homeostasis. In this study, we compared the pharmacological properties of four reported variants of the human GHSR (I134T, V160M, A204E, and F279L) with those of the wild-type receptor. Corresponding recombinant receptors were transiently expressed in either human embryonic kidney 293 or COS-7 cells. Basal as well as ligand-induced signaling was assessed by luciferase reporter gene assays and measurement of inositol phosphate production. In addition, receptor expression levels were monitored by whole-cell enzyme-linked immunosorbent assay. Ligand-independent signaling of the wild-type GHSR is significantly reduced with introduction of either the V160M or F279L substitutions, whereas basal activity of the A204E mutant is not detectable. Ghrelin potency is markedly increased at the V160M mutant, whereas the I134T variant is unresponsive to this endogenous agonist. In contrast, the I134T mutant responds to a known GHSR inverse agonist, [d-Arg1, d-Phe5, d-Trp7,9, Leu11]-substance P (SP-analog), albeit with reduced efficacy. Activity of the SP-analog at the V160M and F279L mutants is comparable to the wild type (WT) value. The overall expression level of each of the four GHSR variants is reduced relative to WT; however, the ratio between the intracellular and plasma membrane receptor density remains comparable. Treatment with the SP-analog significantly increases cell surface expression of each receptor with the exception of the A204E variant. Taken together, our studies reveal that naturally occurring GHSR mutations affect a wide range of pharmacologic properties. The physiological impact of these alterations within selected populations (e.g., obese, lean individuals) as well as the pharmacogenomic consequences of corresponding mutations remain to be further investigated.
Footnotes
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This study was supported by the National Institutes of Health (Grant R01-DK072497 to A.S.K.), by the National Institute of Diabetes and Digestive and Kidney Diseases (Fellowship Grant DK-07542 to G.L.), by the Fonds de la Recherche en Sante du Quebec (postdoctoral fellowship to J.-P.F.), and by the GRASP Digestive Disease Research Center (Grant P30-DK34928).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.123141.
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ABBREVIATIONS: GHSR, growth hormone secretagogue receptor; GPCR, G protein-coupled receptor; SP-analog, [d-Arg1, d-Phe5, d-Trp7,9, Leu11]-substance P; HA, hemagglutinin; BM-blue, 3.3′-5,5′-tetramethylbenzidine; SRE, serum response element; HEK, human embryonic kidney; ELISA, enzyme-linked immunosorbent assay; WT, wild type; PBS, phosphate-buffered saline; IP, inositol phosphate; TM, transmembrane domain; MC4R, melanocortin-4 receptor.
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↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
- Received March 20, 2007.
- Accepted June 26, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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