A Four Amino Acid Deletion Polymorphism in the Third Intracellular Loop of the Human α2C-Adrenergic Receptor Confers Impaired Coupling to Multiple Effectors

doi:10.1074/jbc.M000796200

A Four Amino Acid Deletion Polymorphism in the Third Intracellular Loop of the Human α_2C-Adrenergic Receptor Confers Impaired Coupling to Multiple Effectors*

From the Departments of Medicine and Molecular Genetics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Abstract

The α₂-adrenergic receptors (α₂ARs) play a critical role in modulating neurotransmitter release in the central and peripheral sympathetic nervous systems. A polymorphism of the α₂AR subtype localized to human chromosome 4 (the pharmacologic α_2CAR subtype) within an intracellular domain has been identified in normal individuals. The polymorphism (denoted Del322–325) is because of an in-frame 12-nucleic acid deletion encoding a receptor lacking Gly-Ala-Gly-Pro in the third intracellular loop. To delineate the functional consequences of this structural alteration, Chinese hamster ovary cells were permanently transfected with constructs encoding wild-type human α_2CAR and the polymorphic receptor. The Del322–325 variant had decreased high affinity agonist binding (K_H = 7.3 ± 0.95 versus3.7 ± 0.43 nm; %R_H = 31 ± 4 versus 49 ± 4) compared with wild-type indicating impaired formation of the agonist-receptor-G protein complex. The polymorphic receptor displayed markedly depressed epinephrine-promoted coupling to G_i, inhibiting adenylyl cyclase by 10 ± 4.3% compared with 73 ± 2.4% for wild-type α_2CAR. This also was so for the endogenous ligand norepinephrine and full and partial synthetic agonists. Depressed agonist-promoted coupling to the stimulation of MAP kinase (∼71% impaired) and inositol phosphate production (∼60% impaired) was also found with the polymorphic receptor. The Del322–325 receptor was ∼10 times more frequent in African-Americans compared with Caucasians (allele frequencies 0.381 versus 0.040). Given this significant loss of function phenotype in several signal transduction cascades and the skewed ethnic prevalence, Del322–325 represents a pharmacoethnogenetic locus and may also be the basis for interindividual variation in cardiovascular or central nervous system pathophysiology.

Footnotes

↵* This work was supported in part by National Institutes of Health Grants HL53436, ES06096, and HL41496.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: University of Cincinnati College of Medicine, 231 Bethesda Ave., Rm. 7507, Cincinnati, OH 45267-0564. Tel.: 513-558-4831; Fax: 513-558-0835; E-mail: stephen.liggett@uc.edu.
Published, JBC Papers in Press, May 8, 2000, DOI 10.1074/jbc.M000796200
Abbreviations:

α₂AR

α₂-adrenergic receptor

PCR

polymerase chain reaction

bp

base pair(s)

α_2CAR

α₂AR subtype C

Del322–325

α_2CAR polymorphism resulting in deletion of amino acids 322–325

CHO

Chinese hamster ovary

MAP kinase

mitogen-activated protein kinase

WT

wild-type

GppNHp

5′-guanylylimidodiphosphate
- Received February 2, 2000.
- Revision received April 17, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.