Tyrosine kinase inhibition affects type 1 angiotensin II receptor internalization

Growth factor receptors activate tyrosine kinases and undergo endocytosis. Recent data suggest that tyrosine kinase inhibition can affect growth facto r receptor internalization. The type 1 angiotensin II receptor (AT(1)R) whi ch is a G-protein-coupled receptor, also activates tyrosine kinases and und ergoes endocytosis. Thus, we examined whether tyrosine kinase inhibition af fected AT(1)R internalization. To verify protein tyrosine phosphorylation, both LLCPKCl4 cells expressing rabbit AT(1)R (LLCPKAT1R) and cultured rat m esangial cells (MSC) were treated with angiotensin II (Ang II) [1-100 nM] t hen solubilized and immunoprecipitated with antiphosphotyrosine antisera. I mmunoblots of these samples demonstrated that Ang II stimulated protein tyr osine phosphorylation in both cell types. Losartan [1 mu M], an AT(1)R anta gonist, inhibited Ang II-stimulated protein tyrosine phosphorylation, LLCPK AT1R cells displayed specific I-125-Ang 11 binding at apical (AP) and basol ateral (BL) membranes, and both AP and BL AT(1)R activated tyrosine phospho rylation. LLCPKAT1R cells, incubated with genistein (Gen) [200 mu M] or tyr phostin B-48 (TB-48) [50 mu M], were assayed for acid-resistant specific I- 125-Ang II binding, a measure of Ang TI internalization. Both Gen (n=7) and TB-48 (n=3) inhibited AP I-125-Ang II internalization (80 +/- 7% inhibitio n; p < 0.025 vs. control). Neither compound affected BL internalization. TB -1, a non-tyrosine kinase-inhibiting tyrphostin, did not affect AP I-125-An g 11 endocytosis (n=3), suggesting that the TB-48 effect was specific for t yrosine kinase inhibition. Incubating MSC with Gen (n=5) or herbimycin A [1 50 ng/ml] (n=4) also inhibited MSC I-125-Ang II internalization (82 +/- 11% inhibition; p<0.005 vs. control). Thus, tyrosine kinase inhibition prevent ed Ang IT internalization in MSC and selectively decreased AP Ang II intern alization in LLCPKAT1R cells suggesting that AP AT(1)R in LLCPKATIR cells a nd MSC AT(1)R have similar endocytic phenotypes, and tyrosine kinase activi ty may play a role in AT(1)R internalization.