Role of the N-Terminal Region in G Protein–Coupled Receptor Functions: Negative Modulation Revealed by 5-HT_2BReceptor Polymorphisms

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 HTR 2B gene in a drug-abusing population, we assessed whether these polymorphisms affect 5-hydroxytryptamine 2B (5-HT 2B ) receptor in vitro pharmacologic and coupling properties in transfected COS-7 cells. Modification of the 5-HT 2B 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-HT 2B 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-HT 2B receptor N terminus with a 5-HT 2B receptor bearing a N-terminal deletion, we were able to restore original coupling. This reversion to normal activity of a truncated 5-HT 2B receptor by coexpression of the membrane-tethered wild-type 5-HT 2B receptor N terminus was not observed using a membrane-tethered 5-HT 2B receptor R6G;E42G N terminus. These data suggest that the N terminus exerts a negative control over basal as well as agoniststimulated receptor activity that is lost in the R6G;E42G mutant. Our findings reveal a new and unanticipated role of the 5-HT 2B 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.

Gaddum Memorial Lecture 2014: receptors as an evolving concept: from switches to biased microprocessors

“…While wild‐type receptors will have a natural bias coupling towards signalling proteins, mutation of the receptor will most likely create a different receptor conformation and this, in turn, may change the bias of the mutated receptor towards signalling effectors. In these instances, a natural agonist can be compared with itself in the wild type versus mutant receptor utilizing a synthetic agonist as a reference (to account differences in receptor expression, transduction, etc., see Tschammer et al ., ; Belmer et al ., ). Table shows data on the effects of mutating the dopamine D 2L receptor.…”

Endogenous N-terminal Domain Cleavage Modulates α1D-Adrenergic Receptor Pharmacodynamics

“…Prototypical examples are the metabotropic glutamate receptors, which use their extremely long NT to form the glutamate-binding site and modulate ligand selectivity (41). A double mutant polymorphism (R6G/E42G) in the 5HT2B serotonin receptor NT identified in drug-abuse patients (42) was found to negatively modulate both basal and agonist-stimulated receptor activity (43). A similar mechanism has been demonstrated for the H1 histamine receptor, where NT domain association with extracellular loop 2 imparts distinct pharmacological properties to the receptor (44).…”

Buprenorphine signalling is compromised at the N40D polymorphism of the human μ opioid receptor in vitro

“…N-terminal polymorphisms affect signalling in other related GPCRs. N-terminal single nucleotide polymorphisms (SNPs) in the 5-HT2B receptor increased constitutive and agonist-stimulated activity in COS-7 cells (Belmer et al, 2014). Likewise, polymorphisms of the N-terminal region in the melanocortin MC4 receptor increased constitutive activity (Srinivasan et al, 2004).…”