REGIONS OF THE ALPHA-1-ADRENERGIC RECEPTOR INVOLVED IN COUPLING TO PHOSPHATIDYLINOSITOL HYDROLYSIS AND ENHANCED SENSITIVITY OF BIOLOGICAL FUNCTION

Regions of the hamster α1-adrenergic receptor (α1AR) that are important in GTP-binding protein (G protein)-mediated activation of phospholipase C were determined by studying the biological functions of mutant receptors constructed by recombinant DNA techniques. A chimeric receptor consisting of the β2-adrenergic receptor (β2AR) into which the putative third cytoplasmic loop of the α1AR had been placed activated phosphatidylinositol metabolism as effectively as the native α1AR, as did a truncated α1AR lacking the last 47 residues in its cytoplasmic tail. Substitutions of β2AR amino acid sequence in the intermediate portions of the third cytoplasmic loop of the α1AR or at the N-terminal portion of the cytoplasmic tail caused marked decreases in receptor coupling to phospholipase C. Conservative substitutions of two residues in the C terminus of the third cytoplasmic loop (Ala293 → Leu, Lys290 → His) increased the potency of agonists for stimulating phosphatidylinositol metabolism by up to 2 orders of magnitude. These data indicate (i) that the regions of the α1AR that determine coupling to phosphatidylinsotiol metabolism are similar to those previously shown to be involved in coupling of β2AR to adenylate cyclase stimulation and (ii) that point mutations of a G-protein-coupled receptor can cause remarkable increases in sensitivity of biological response.