title: Side-chain substitutions within angiotensin II reveal different requirements for signaling, internalization, and phosphorylation of type 1A angiotensin receptors.
abstract: Binding of the peptide hormone angiotensin II (AngII) to the type 1 (AT(1A)) receptor and the subsequent activation of phospholipase C-mediated signaling, involves specific determinants within the AngII peptide sequence. In contrast, the contribution of such determinants to AT(1A) receptor internalization, phosphorylation and activation of mitogen-activated protein kinase (MAPK) signaling is not known. In this study, the internalization of an enhanced green fluorescent protein-tagged AT(1A) receptor (AT(1A)-EGFP), in response to AngII and a series of substituted analogs, was visualized and quantified using confocal microscopy. AngII-stimulation resulted in a rapid, concentration-dependent internalization of the chimeric receptor, which was prevented by pretreatment with the nonpeptide AT(1) receptor antagonist EXP3174. Remarkably, AT(1A) receptor internalization was unaffected by substitution of AngII side chains, including single and double substitutions of Tyr(4) and Phe(8) that abolish phospholipase C signaling through the receptor. AngII-induced receptor phosphorylation was significantly inhibited by several substitutions at Phe(8) as well as alanine replacement of Asp(1). The activation of MAPK was only significantly inhibited by substitutions at position eight in the peptide and specific substitutions did not equally inhibit inositol phosphate production, receptor phosphorylation and MAPK activation. These results indicate that separate, yet overlapping, contacts made between the AngII peptide and the AT(1A) receptor select/induce distinct receptor conformations that preferentially affect particular receptor outcomes. The requirements for AT(1A) receptor internalization seem to be less stringent than receptor activation and signaling, suggesting an inherent bias toward receptor deactivation.
authors.affiliation: Molecular Endocrinology Laboratory, Baker Medical Research Institute, Melbourne, Australia.
authors.forename: Alice C
authors.initials: AC
authors.lastname: Holloway
authors.forename: Hongwei
authors.initials: H
authors.lastname: Qian
authors.forename: Luisa
authors.initials: L
authors.lastname: Pipolo
authors.forename: James
authors.initials: J
authors.lastname: Ziogas
authors.forename: Shin-ichiro
authors.initials: S
authors.lastname: Miura
authors.forename: Sadashiva
authors.initials: S
authors.lastname: Karnik
authors.forename: Bridget R
authors.initials: BR
authors.lastname: Southwell
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Lew
authors.forename: Walter G
authors.initials: WG
authors.lastname: Thomas
mesh_terms: D000804:Angiotensin II / Q000737:chemistry / Q000502:physiology*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D010766:Phosphorylation; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D008164:Luminescent Proteins; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D000804:Angiotensin II; D049452:Green Fluorescent Proteins; D010649:Phenylalanine; D020929:Mitogen-Activated Protein Kinase Kinases