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 angioten sin 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)) recept ors. We determined signaling pathways for ANG IV in a proximal tubular cell line, LLC-PK1/Cl-4. In these cells, we found no specific binding of [I-125 ]-ANG II. In contrast, ANG IV dose dependently competed for [I-125]-labeled ANG IV binding, with no displacement by either ANG II, the AT1 receptor an tagonist losartan, or the AT(2) antagonist PD-123319. Saturation binding in dicated the presence of AT4 receptors of high affinity [dissociation consta nt (K-d) = 1.4 nM]. ANG IV did not affect cAMP or cGMP production and did n ot increase cytosolic calcium concentration in these cells. In contrast, im munoprecipitation and immunoblotting studies revealed that ANG IV caused do se-dependent tyrosine phosphorylation of p125-focal adhesion kinase (p125-F AK) and p68-paxillin within 2 min, with maximal stimulation at 30 min. ANG IV-stimulated tyrosine phosphorylation of p125-FAK and paxillin was not aff ected by pretreatment with either losartan or PD-123319, and ANG II (10(-7) M) did not induce protein tyrosine phosphorylation. Our results indicate t hat LLC-PK1/Cl-4 cells express ANG IV receptors, which we demonstrate for t he first time are linked to tyrosine phosphorylation of focal adhesion-asso ciated proteins. This suggests that ANG IV, a product of ANG II metabolism, may regulate function of the focal adhesion complex in proximal tubule cel ls.