title: The tyrosine within the NPXnY motif of the human angiotensin II type 1 receptor is involved in mediating signal transduction but is not essential for internalization.
abstract: The NPXnY motif is involved in the internalization process of several types of receptors, including lipoprotein receptors and G protein-coupled receptors. We replaced Tyr302 with either phenylalanine or alanine in the NPLFY site of the human angiotensin II receptor type 1 and determined the pharmacological properties of the resulting mutant receptors. Competitive binding experiments revealed that COS-7 cells transfected with either the wild-type or mutant receptors expressed approximately the same amount of high affinity binding sites (Bmax 70,000 sites/cell and Kd approximately 2 nM). Photoaffinity labeling of both native and mutant receptors revealed apparent molecular masses of 110 kDa. Incubation of transfected cells with 0.2 nM [125I]Ang II at 37 degrees revealed an efficient internalization of the wild-type receptor and the mutant receptors, although the mutant receptors were internalized at a slower rate. Interestingly, however, the transmembrane signaling was severely impaired in transfected cells expressing mutant receptors. No significant production of inositol-1,4,5-trisphosphate was observed when these cells were challenged for 3 min with a concentration of angiotensin II as high as 1 microM. This is in contrast to the dose-dependent stimulation of inositol-1,4,5-trisphosphate production in cells expressing the wild-type receptor. Thus, our results show that the Tyr302 in the NPXnY motif of the human angiotensin II receptor type 1 is not essential for agonist binding properties or for internalization of the receptor but plays an important role in transmembrane signaling.
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: G
authors.initials: G
authors.lastname: Servant
authors.forename: D E
authors.initials: DE
authors.lastname: Richard
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: D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004705:Endocytosis; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011945:Receptors, Angiotensin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D017931:DNA Primers; D011945:Receptors, Angiotensin; D014443:Tyrosine; D010738:Type C Phospholipases