Gbeta gamma -independent constitutive association of Galpha s with SHP-1 and angiotensin II receptor AT2 is essential in AT2-mediated ITIM-independent activation of SHP-1.
Conventional mode of activation of SH2 domain-containing phosphatase 1 (SHP-1) by a single transmembrane (TM) inhibitory receptor such as killer cell inhibitory receptor, Fcgamma receptor type IIb1, and paired Ig-like receptors of inhibitory types requires tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory (ITIM) motifs in the cytoplasmic domains of the inhibitory receptors. Contrary to this paradigm, AT(2), a G protein-coupled 7TM receptor that does not undergo tyrosine phosphorylation in response to angiotensin II (Ang II) stimulation, also activates SHP-1. Here we show that SHP-1 constitutively and physically associates with AT(2) receptor in transfected COS-7 cells. On stimulation by Ang II, SHP-1 becomes activated and dissociated from AT(2) receptor, independent of pertussis toxin. Cotransfection of transducin G(betagamma) inhibits SHP-1/AT(2) association and the SHP-1 activation, whereas cotransfection of C-terminal of beta-adrenergic receptor kinase, which abrogates G(betagamma) signaling, facilitates SHP-1 activation. Surprisingly, SHP-1/AT(2) association and the SHP-1 activation requires the presence of G(alphas) as shown by differential coimmunoprecipitation, dominant negative G(alphas), constitutively active G(alphas), and G(alpha) peptides. A mutant AT(2) receptor D141A-R142L that is inactive in G(alpha) protein activation constitutively associates with SHP-1 and activates it. Together, these results indicate that G(alphas) alone, rather than exclusively in the form of G(alphabetagamma) heterotrimer may facilitate signal transduction for G protein-coupled receptors, suggesting a novel mechanism distinct from the classic paradigm of heterotrimeric G proteins. The AT(2)-mediated ITIM-independent activation of SHP-1 that is distinct from the conventional mode of activation, may represent a general paradigm for activation of SHP-1/2-class tyrosine phosphatases by G protein-coupled receptors.
Feng
Ying-Hong
YH
Department of Medicine, Case Western Reserve University School of Medicine, and University Hospital of Cleveland, 10900 Euclid Avenue, Cleveland, OH 44106-4982, USA. yxf6@po.cwru.edu
Sun
Yan
Y
Douglas
Janice G
JG
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D009154:Mutation; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D051552:beta-Adrenergic Receptor Kinases
C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D047908:Intracellular Signaling Peptides and Proteins; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D054645:Protein Phosphatase 1; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases; C509250:Ptpn6 protein, rat; D020962:Heterotrimeric GTP-Binding Proteins