G(βγ)-independent constitutive association of G(αs) with SHP-1 and angiotensin II receptor AT(2) is essential in AT(2)-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, Fcγ 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(βγ) inhibits SHP-1/AT(2) association and the SHP-1 activation, whereas cotransfection of C-terminal of β-adrenergic receptor kinase, which abrogates G(βγ) signaling, facilitates SHP-1 activation. Surprisingly, SHP-1/AT(2) association and the SHP-1 activation requires the presence of G(αs) as shown by differential coimmunoprecipitation, dominant negative G(αs), constitutively active G(αs), and G(α) peptides. A mutant AT(2) receptor D141A–R142L that is inactive in G(α) protein activation constitutively associates with SHP-1 and activates it. Together, these results indicate that G(αs) alone, rather than exclusively in the form of G(αβγ) 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.