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Tyrosine kinase inhibition affects type 1 angiotensin II receptor internalization
B N Becker1, S Kondo, J K Chen
1Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Abstract:
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 LLCPKCl4 cells expressing rabbit AT1R (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 microM], an AT1R antagonist, inhibited Ang II-stimulated protein tyrosine phosphorylation. LLCPKAT1R cells displayed specific 125I-Ang II binding at apical (AP) and basolateral (BL) membranes, and both AP and BL AT1R activated tyrosine phosphorylation. LLCPKAT1R cells, incubated with genistein (Gen) [200 microM] or tyrphostin B-48 (TB-48) [50 microM], 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 LLCPKAT1R cells suggesting that AP AT1R in LLCPKAT1R cells and MSC AT1R have similar endocytic phenotypes, and tyrosine kinase activity may play a role in AT1R internalization.
Insights
Tyrosine kinase inhibition significantly reduced angiotensin II (Ang II) internalization in mesangial cells and selectively decreased apical AT1R internalization. This suggests tyrosine kinase activity plays a role in AT1R endocytosis.
Area of Science:
- Cell Biology
- Biochemistry
- Pharmacology
Background:
- Growth factor receptors activate tyrosine kinases and undergo endocytosis.
- Recent data suggest tyrosine kinase inhibition affects growth factor receptor internalization.
- The type 1 angiotensin II receptor (AT1R), a G-protein-coupled receptor, activates tyrosine kinases and undergoes endocytosis.
Purpose of the Study:
- To investigate whether tyrosine kinase inhibition affects AT1R internalization.
- To determine the role of tyrosine kinase activity in AT1R endocytosis.
Main Methods:
- LLCPKCl4 cells expressing rabbit AT1R and cultured rat mesangial cells (MSC) were treated with angiotensin II (Ang II).
- Protein tyrosine phosphorylation was assessed via immunoprecipitation and immunoblotting.
- Ang II internalization was measured using 125I-Ang II binding assays after treatment with tyrosine kinase inhibitors (genistein, tyrphostin B-48, herbimycin A) or a control (TB-1).
Main Results:
- Ang II stimulated protein tyrosine phosphorylation in both LLCPKAT1R and MSC.
- Losartan, an AT1R antagonist, inhibited Ang II-stimulated tyrosine phosphorylation.
- Genistein and tyrphostin B-48 significantly inhibited apical (AP) 125I-Ang II internalization in LLCPKAT1R cells (80% inhibition).
- Neither genistein nor herbimycin A affected basolateral (BL) internalization.
- Genistein and herbimycin A inhibited 125I-Ang II internalization in MSC (82% inhibition).
- The non-tyrosine kinase-inhibiting tyrphostin B-1 did not affect AP 125I-Ang II endocytosis.
Conclusions:
- Tyrosine kinase inhibition prevented Ang II internalization in MSC.
- Tyrosine kinase inhibition selectively decreased AP Ang II internalization in LLCPKAT1R cells.
- AP AT1R in LLCPKAT1R cells and MSC AT1R exhibit similar endocytic phenotypes.
- Tyrosine kinase activity appears to play a role in AT1R internalization.