title: Essential role of leucine222 in mediating signal transduction of the human angiotensin II type 1 receptor.
abstract: The angiotensin II (Ang II) type I receptor (AT1) is a member of the superfamily of heptahelical, G protein-coupled receptors (GPCRs) characterized by hydrophobic transmembrane domains connected by extra- and intracellular hydrophilic loops. The third intracellular loop (IC3) of many GPCRs is thought to directly interact with G proteins. We examined the molecular environment of the basic sequence KA221L222KK found in the IC3 of the human AT1 (hAT1) receptor by substituting Ala221 for Glu/Gln or Leu222 for Arg/Gln and determined the pharmacological properties of the resulting mutant receptors. Competitive binding experiments with the antagonist [Sar1,Ile8]Ang II revealed that COS-7 or stably expressing CHO cells transfected with either the wild-type or mutant receptors, produced a single population of high affinity binding sites (Kd of 0.5 nM) but variable receptor levels depending on cell type; Bmax approximately 100,000 sites/cell (COS-7), approximately 20,000 sites/cell (CHO). However, in competitive binding experiments using the agonist Ang II, both the wild type and the Ala-->Glu mutant displayed binding affinities of 1 nM, while the Leu-->Arg mutant had a significantly lower affinity (4 nM). When the functionality of the mutant receptors was examined, a lowered production of inositol-1,4,5-trisphosphate was obtained upon stimulation of the Ala-->Glu and Ala-->Gln mutants when compared to the wild-type receptor. However, no significant production of Ins(1,4,5)P3 was detected for the Leu-->Arg and leu-->Gln mutants. Our results suggest that Leu222 in the KALKK sequence of the IC3 of the hAT1 receptor, through not essential for antagonist binding, has an essential role in mediating interactions with G protein and in signal transduction.
authors.affiliation: Department of Pharmacology, Faculty of Medicine, Université de Sherbrooke, Quebec, Canada.
authors.forename: S A
authors.initials: SA
authors.lastname: Laporte
authors.forename: S F
authors.initials: SF
authors.lastname: Roy
authors.forename: E
authors.initials: E
authors.lastname: Escher
authors.forename: G
authors.initials: G
authors.lastname: Guillemette
authors.forename: R
authors.initials: R
authors.lastname: Leduc
mesh_terms: D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D004789:Enzyme Activation; D006801:Humans; D007930:Leucine / Q000235:genetics / Q000502:physiology*; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D010738:Type C Phospholipases; D007930:Leucine