Alvarez Sergio E, Seguin Leonardo R, Villarreal Rodrigo S, Nahmias Clara, Ciuffo Gladys M
Facultad de Química, Instituto Multidisciplinario de Investigaciones Biológicas (IMIBIO-CONICET), Bioquímica y Farmacia, Universidad Nacional de San Luis, San Luis, Argentina.
J Cell Biochem. 2008 Oct 15;105(3):703-11. doi: 10.1002/jcb.21866.
Angiotensin II (Ang II) AT(2) receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10(-7) M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT(2) antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT(2) receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT(2) or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT(2) or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT(2) receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT(2) receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT(2) subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.
血管紧张素II（Ang II）AT(2)受体在大鼠胎儿组织中大量表达，可能对发育有促进作用。在本研究中，我们检测了Ang II 2型受体刺激对SHP-1激活的影响。Ang II（10(-7) M）以剂量依赖的形式引发SHP-1快速且短暂的酪氨酸磷酸化，在1分钟时达到最大值，可被AT(2)拮抗剂PD123319阻断。SHP-1磷酸化之后不同蛋白质的酪氨酸去磷酸化随之发生，提示一系列事件的发生顺序。如共免疫沉淀、蛋白质印迹和结合试验所示，Ang II诱导SHP-1与AT(2)受体结合。在Ang II刺激（1分钟）后，用抗AT(2)或抗SHP-1抗体获得免疫复合物，测定SHP-1活性，并与SHP-1磷酸化的最大水平相关联。有趣的是，受体刺激（1分钟）后，c-Src与AT(2)或SHP-1免疫复合物结合。用c-Src抑制剂PP2预孵育可抑制SHP-1激活和c-Src结合，从而证实c-Src参与此途径。我们首次在此处证明，在同时表达两种受体亚型的离体模型中，c-Src通过AT(2)受体参与SHP-1激活。在此模型中，AT(2)受体并非组成性地与SHP-1结合，且SHP-1也未组成性激活。因此，我们明确证实，在大鼠胎膜中，由AT(2)亚型介导的SHP-1激活涉及c-Src，且先于蛋白质酪氨酸去磷酸化。
