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 (AT1R) which is a G-protein-coupled receptor, also activates tyrosine kinases and undergoes endocytosis. Thus, we examined whether tyrosine kinase inhibition affected AT1R internalization. To verify protein tyrosine phosphorylation, both LLCPKCI₄ cells expressing rabbit AT1R (LLCPK(AT)1(R)) 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 μM], an AT1R antagonist, inhibited Ang II-stimulated protein tyrosine phosphorylation. LLCPK(AT1R) cells displayed specific ¹²⁵I-Ang II binding at apical (AP) and basolateral (BL)membranes, and both AP and BL AT1R activated tyrosine phosphorylation. LLCPK(AT)1(R) cells, incubated with genistein (Gen) [200 μM] or tyrphostin B-48 (TB-48) [50 μM], were assayed for acid-resistant specific ¹²⁵I-Ang II binding, a measure of Ang II internalization. Both Gen (n=7) and TB-48 (n=3) inhibited AP ¹²⁵I-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 ¹²5I-Ang II 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 ¹²5I-Ang II internalization (82 ± 11% inhibition; p<0.005 vs. control). Thus, tyrosine kinase inhibition prevented Ang II internalization in MSC and selectively decreased AP Ang II internalization in LLCPK(AT1R) cells suggesting that AP AT1R in LLCPK(AT1R) cells and MSC AT1R have similar endocytic phenotypes, and tyrosine kinase activity may play a role in AT1R internalization.