Dependence on the Motif YIPP for the Physical Association of Jak2 Kinase with the Intracellular Carboxyl Tail of the Angiotensin II AT1 Receptor

Abstract

Angiotensin II is the effector molecule of the reninangiotensin system. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT 1 . These receptors contain the structural features of the seven-transmembrane, G-proteincoupled receptor superfamily. Angiotensin II, acting through the AT 1 receptor, also stimulates the Jak/STAT pathway by inducing ligand-dependent Jak2 tyrosine phosphorylation and activation. Here, we show that a glutathione S-transferase fusion protein containing the carboxyl-terminal 54 amino acids of the rat AT 1A receptor physically binds to Jak2 in an angiotensin II-dependent manner. Deletional analysis, using both in vitro protocols and cell transfection analysis, showed that this association is dependent on the AT 1A receptor motif YIPP (amino acids 319 -322). The wild-type AT 1A receptor can induce Jak2 tyrosine phosphorylation. In contrast, an AT 1A receptor lacking the YIPP motif is unable to induce ligand-dependent phosphorylation of Jak2. Competition experiments with synthetic peptides suggest that each of the YIPP amino acids, including tyrosine 319, is important in Jak2 binding to the AT 1A receptor. The binding of the AT 1A receptor to the intracellular tyrosine kinase Jak2 supports the concept that the seven-transmembrane superfamily of receptors can physically associate with enzymatically active intracellular proteins, creating a signaling complex mechanistically similar to that observed with growth factor and cytokine receptors.The analysis of cytokines and their receptors has implicated the intracellular Jak family of kinases as critically important for the intracellular signaling initiated in response to ligand (1-3). Cytokines induce receptor dimerization and the activation, via tyrosine phosphorylation, of the associated Jak kinases. The Jak kinases phosphorylate the cytokine receptors, leading to the binding and eventual activation of intermediate signaling molecules referred to as STAT (signal transducers and activators of transcription). The STAT proteins are a family of transcription factors that migrate to the nucleus and induce gene transcription (4). The Jak/STAT pathway was first elucidated through the study of interferon signaling, but it is now known that this pathway participates in the signaling initiated by a wide variety of cytokines and growth factors. Recently, the vasoactive peptide angiotensin II was also found to activate the Jak/STAT pathway (5).Angiotensin II is the effector molecule of the renin-angiotensin system. It is an 8-amino acid peptide that induces several physiologic responses that act to raise blood pressure. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT 1 (6). Whereas humans have a single AT 1 receptor gene, rodents possess two genes encoding highly homologous receptor isoforms termed AT 1A and AT 1B . These proteins are 95% identical and appear to bind ligand and to signal in an identical fashion (7,8). All AT 1 receptors...

The angiotensin II type 1 receptor and receptor-associated proteins

“…After the initial report of induction of the JAK-STAT pathway by the AT1 receptor, Marrero et al [38] demonstrated that it interacts directly with JAK2 kinase, providing the mechanism of JAK-STAT activation. Subsequently, they proposed that JAK2 is associated with the AT1A receptor through the Tyr319Ile320Pro321Pro322 (YIPP, Fig 3) sequence within the proximal carboxyl-terminal region of AT1A receptor [59]. While this YIPP motif may function as a SH2 targeting sequence upon phosphorylation of Tyr319, JAK2 contains no SH2 domains that could putatively mediate the association.…”

Angiotensin II-induced Association of Phospholipase Cγ1 with the G-protein-coupled AT1 Receptor

“…1 The abbreviations used are: SH2, Src homology 2; PLC, phospholipase C; Ang II, angiotensin II; VSMC, vascular smooth muscle cells; IP 3 , inositol 1,4,5-trisphosphate; PIP 2 , phosphatidylinositol 4,5-bisphosphate; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-tyrosine phosphorylation, activation, and association of JAK2 with the receptor (10). JAK2-receptor association appears to depend on a YIPP motif in the C-terminal intracellular domain of the AT 1 receptor that is identical to the PLC␥1 SH2 domain binding site identified in the platelet-derived growth factor receptor (11). Because JAK2 does not contain any SH2 domains, the finding that JAK2 associates with this motif in the AT 1 receptor was initially puzzling.…”

“…Point mutations and deletional mutations were introduced into the constructs as described previously (11). The sequences of all DNA constructs were verified by DNA sequence analysis.…”

Angiotensin II receptor pharmacology and AT1-receptor blockers

“…68 A physical association between Jak2 kinase and a part of the intracellular carboxy tail of the AT 1 receptor has been shown. 69 Very recently it has been shown that Ang II mediates the Jak/STAT pathway under pathological conditions. 70 In contrast to the AT 1 receptor, much less is known about the structural and functional properties of the AT 2 receptor, which has been cloned recently from the mouse, rat, and human.…”