Angiotensin IV induces tyrosine phosphorylation of focal adhesion kinase and paxillin in proximal tubule cells.
Angiotensin IV (ANG IV), the COOH-terminal hexapeptide fragment of angiotensin II (ANG II), binds to specific sites in the kidney, distinct from type 1 (AT(1)) and type 2 (AT(2)) receptors and designated type 4 (AT(4)) receptors. We determined signaling pathways for ANG IV in a proximal tubular cell line, LLC-PK(1)/Cl(4). In these cells, we found no specific binding of [(125)I]-ANG II. In contrast, ANG IV dose dependently competed for [(125)I]-labeled ANG IV binding, with no displacement by either ANG II, the AT(1) receptor antagonist losartan, or the AT(2) antagonist PD-123319. Saturation binding indicated the presence of AT(4) receptors of high affinity [dissociation constant (K(d)) = 1.4 nM]. ANG IV did not affect cAMP or cGMP production and did not increase cytosolic calcium concentration in these cells. In contrast, immunoprecipitation and immunoblotting studies revealed that ANG IV caused dose-dependent tyrosine phosphorylation of p125-focal adhesion kinase (p125-FAK) and p68-paxillin within 2 min, with maximal stimulation at 30 min. ANG IV-stimulated tyrosine phosphorylation of p125-FAK and paxillin was not affected by pretreatment with either losartan or PD-123319, and ANG II (10(-7) M) did not induce protein tyrosine phosphorylation. Our results indicate that LLC-PK(1)/Cl(4) cells express ANG IV receptors, which we demonstrate for the first time are linked to tyrosine phosphorylation of focal adhesion-associated proteins. This suggests that ANG IV, a product of ANG II metabolism, may regulate function of the focal adhesion complex in proximal tubule cells.
Chen
J K
JK
Division of Nephrology, Department of Medicine, Kidney Research Center, Ottawa Hospital Research Institute, University of Ottawa, Ottawa, Ontario, Canada K1H 8L6.
Zimpelmann
J
J
Harris
R C
RC
Burns
K D
KD
D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D051416:Focal Adhesion Protein-Tyrosine Kinases; D007457:Iodine Radioisotopes; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000201:enzymology*; D018374:LLC-PK1 Cells; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D012084:Renin-Angiotensin System / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013552:Swine; D013936:Thymidine / Q000493:pharmacokinetics; D014316:Tritium; D014443:Tyrosine / Q000378:metabolism
D003598:Cytoskeletal Proteins; D007457:Iodine Radioisotopes; D051419:Paxillin; D010750:Phosphoproteins; D014316:Tritium; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-; D014443:Tyrosine; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006152:Cyclic GMP; D002118:Calcium; D013936:Thymidine