Abstract
The putative role of the N-terminal region of rhodopsin-like 7 transmembrane biogenic amine receptors in agonist-induced signaling has not yet been clarified despite recent advances in 7 transmembrane receptor structural biology. Given the existence of N-terminal nonsynonymous polymorphisms (R6G;E42G) within the HTR2B gene in a drug-abusing population, we assessed whether these polymorphisms affect 5-hydroxytryptamine 2B (5-HT2B) receptor in vitro pharmacologic and coupling properties in transfected COS-7 cells. Modification of the 5-HT2B receptor N terminus by the R6G;E42G polymorphisms increases such agonist signaling pathways as inositol phosphate accumulation as assessed by either classic or operational models. The N-terminal R6G;E42G mutations of the 5-HT2B receptor also increase cell proliferation and slow its desensitization kinetics compared with the wild-type receptor, further supporting a role for the N terminus in transduction efficacy. Furthermore, by coexpressing a tethered wild-type 5-HT2B receptor N terminus with a 5-HT2B receptor bearing a N-terminal deletion, we were able to restore original coupling. This reversion to normal activity of a truncated 5-HT2B receptor by coexpression of the membrane-tethered wild-type 5-HT2B receptor N terminus was not observed using a membrane-tethered 5-HT2B receptor R6G;E42G N terminus. These data suggest that the N terminus exerts a negative control over basal as well as agonist-stimulated receptor activity that is lost in the R6G;E42G mutant. Our findings reveal a new and unanticipated role of the 5-HT2B receptor N terminus as a negative modulator, affecting both constitutive and agonist-stimulated activity. Moreover, our data caution against excluding the N terminus and extracellular loops in structural studies of this 7 transmembrane receptor family.
Footnotes
- Received August 9, 2013.
- Accepted October 30, 2013.
↵1 Current affiliation: Department of Physiology and Pharmacology, Robert C. Byrd Health Sciences Center, School of Medicine, West Virginia University, Morgantown, West Virginia.
A.B. and S.D. contributed equally to this work.
This work was supported by the Centre National de la Recherche Scientifique; the Institut National de la Santé et de la Recherche Médicale; the Université Pierre et Marie Curie; by grants from the Fondation de France; the French Ministry of Research Agence Nationale pour la Recherche [ANR-12-BSV1-0015-01]; a Lefoulon-Lalande fellowship (to S.D.); a Société Française de Pharmacologie Thérapeutique fellowship (to A.B.); and a Marie Curie EU fellowship (to V.S.). L.M.’s team is part of the École des Neurosciences de Paris Ile-de-France network and of the Bio-Psy Labex.
↵This article has supplemental material available at molpharm.aspetjournals.org.
- Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics
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