Identification of a Gs Coupling Domain in the Amino Terminus of the Third Intracellular Loop of the α2A-Adrenergic Receptor

作者：Margaret G. Eason, Stephen B. Liggett · 发表于：Journal of Biological Chemistry · 年份：1995 · DOI：10.1074/jbc.270.42.24753 · 被引用次数：108 · 研究领域：Receptor Mechanisms and Signaling、Protein Kinase Regulation and GTPase Signaling、Neuropeptides and Animal Physiology

alpha2-Adrenergic receptors (alpha 2AR) functionally couple not only to Gi but also to Gs. We investigated the amino-terminal portion of the third intracellular loop of the human alpha 2AAR (alpha 2C10) for potential Gs coupling domains using site-directed mutagenesis and recombinant expression in several different cell types. A deletion mutant and four chimeric receptors consisting of the alpha 2AAR with the analogous sequence from the 5-HT1A receptor (a Gi-coupled receptor) and the beta 2AR (a Gs-coupled receptor) were expressed in Chinese hamster ovary cells, Chinese hamster fibroblasts, or COS-7 cells and examined for their ability to mediate stimulation or inhibition of membrane adenylyl cyclase activity or whole cell cAMP accumulation. In stably expressing Chinese hamster ovary cells, deletion of amino acids 221-231, which are in close proximity to the fifth transmembrane domain, eliminated alpha 2C10-mediated stimulation of adenylyl cyclase activity, while alpha 2C10-mediated inhibition was only moderately affected. This suggested that this region is important for Gs coupling, prompting construction of the chimeric receptor mutants. Substitution of amino acids 218-235 with 5-HT1A receptor sequence entirely ablated agonist-promoted Gs coupling, as compared with a 338 +/- 29% stimulation of adenylyl cyclase activity observed with the wild-type alpha 2C10. In contrast, Gi coupling for this mutant remained fully intact (57 +/- 2% versus 52 +/- 1% inhibition for wild-type a...