SUSTAINED EFFECT OF ANGIOTENSIN-II ON TYROSINE PHOSPHORYLATION OF ANNEXIN-I IN GLOMERULAR MESANGIAL CELLS

By means of selective extraction in a Ca2+-chelating medium and immuno blotting, four annexins (I, II, V, and VI) were identified in both iso lated rat renal glomeruli and rat glomerular mesangial cells. Upon P-3 2 labeling of these cells in culture, annexin I was immunoprecipitated using a specific polyclonal antibody and was found to incorporate rad ioactivity in a constitutive manner. However, as with epidermal growth factor (200 ng/ml), addition of angiotensin II (10(-7) M), arginine-v asopressin (10(-7) M), or endothelin I (10(-7) M) resulted in a 2-3-fo ld stimulation of annexin I phosphorylation. The basal phosphorylation as well as the stimulating effect of angiotensin II were also detecte d by immunoblotting annexin extracts using an anti-phosphotyrosine ant ibody. In addition, among various phosphotyrosyl proteins isolated fro m EGTA extracts by adsorption onto an anti-phosphotyrosine antibody, a nnexin I was specifically recognized by Western blotting using a monoc lonal anti-annexin I antibody, and displayed the same increase upon ce ll stimulation with angiotensin II. Moreover, thin layer chromatograph ic analysis of phosphoamino acids present in immunoprecipitated [P-32] annexin I showed an exclusive labeling of phosphotyrosine residue(s). Finally, the effect of angiotensin II was detectable after 10 min, max imal at 6 h, and present until 12 h of incubation. Using 12-h stimulat ion, tyrosine phosphorylation of annexin I displayed a maximum at 10(- 7) to 10(-6) M angiotensin II. These data report for the first time th e stimulation of annexin I tyrosine phosphorylation by biologically ac tive peptides acting via receptors belonging to the superfamily of sev en hydrophobic domain, G-protein-linked receptors, which lack an intri nsic protein tyrosine kinase. This suggests a possible role of annexin I in the mitogenic effect of angiotensin II, arginine-vasopressin, an d endothelin I, which was previously observed on rat glomerular mesang ial cells as well as on other cells.