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

摘要

Conventional mode of activation of SH2 domain-containing phos-phatase 1 (SHP-1) by a single transmembrane (TM) inhibitory receptor such as killer cell inhibitory receptor, F_(cγ) receptor type Ilb1, and paired lg-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 con-stitutively and physically associates with AT_2 receptor in transfected COS-7 cells. On stimulation by Ang II, SHP-1 becomes activated and dissociated from AT2 receptor, independent of pertussis toxin. Co-transfection of transducin G_(βγ) inhibits SHP-1/AT2 association and the SHP-1 activation, whereas cotransfection of C-terminal of β-adrener-gic 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 coimmu-noprecipitation, 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.