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 factor receptor internalization. The type 1 angiotensin II receptor (AT(1)R) which is a G-protein-coupled receptor, also activates tyrosine kinases and undergoes endocytosis. Thus, we examined whether tyrosine kinase inhibition affected AT(1)R internalization. To verify protein tyrosine phosphorylation, both LLCPKCl4 cells expressing rabbit AT(1)R (LLCPKAT1R) and cultured rat mesangial cells (MSC) were treated with angiotensin II (Ang II) [1-100 nM] then solubilized and immunoprecipitated with antiphosphotyrosine antisera. Immunoblots of these samples demonstrated that Ang II stimulated protein tyrosine phosphorylation in both cell types. Losartan [1 mu M], an AT(1)R antagonist, inhibited Ang II-stimulated protein tyrosine phosphorylation, LLCPKAT1R cells displayed specific I-125-Ang 11 binding at apical (AP) and basolateral (BL) membranes, and both AP and BL AT(1)R activated tyrosine phosphorylation. LLCPKAT1R cells, incubated with genistein (Gen) [200 mu M] or tyrphostin 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% inhibition; p < 0.025 vs. control). Neither compound affected BL internalization. TB-1, a non-tyrosine kinase-inhibiting tyrphostin, did not affect AP I-125-Ang 11 endocytosis (n=3), suggesting that the TB-48 effect was specific for tyrosine kinase inhibition. Incubating MSC with Gen (n=5) or herbimycin A [150 ng/ml] (n=4) also inhibited MSC I-125-Ang II internalization (82 +/- 11% inhibition; p<0.005 vs. control). Thus, tyrosine kinase inhibition prevented Ang IT internalization in MSC and selectively decreased AP Ang II internalization in LLCPKAT1R cells suggesting that AP AT(1)R in LLCPKATIR cells and MSC AT(1)R have similar endocytic phenotypes, and tyrosine kinase activity may play a role in AT(1)R internalization.