Angiotensin IV induces tyrosine phosphorylation of focal adhesion kinase and paxillin in proximal tubule cells

Jiankang Chen, Joe Zimpelmann, Raymond C. Harris, Kevin D. Burns

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

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 (AT1) and type 2 (AT2) receptors and designated type 4 (AT4) receptors. We determined signaling pathways for ANG IV in a proximal tubular cell line, LLC-PK1/Cl4. In these cells, we found no specific binding of [125I]-ANG II. In contrast, ANG IV dose dependently competed for [125I]-labeled ANG IV binding, with no displacement by either ANG II, the AT1 receptor antagonist losartan, or the AT2 antagonist PD-123319. Saturation binding indicated the presence of AT4 receptors of high affinity [dissociation constant (Kd) = 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-PK1/Cl4 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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Renal Physiology
Volume280
Issue number6 49-6
StatePublished - Jul 4 2001
Externally publishedYes

Fingerprint

Paxillin
Focal Adhesion Protein-Tyrosine Kinases
Tyrosine
Phosphorylation
Angiotensin II
Focal Adhesions
Losartan
LLC-PK1 Cells
Angiotensin Type 1 Receptor
des-Asp(1)-des-Arg(2)-Ile(5)-angiotensin II
Angiotensin Receptor Antagonists
Immunoprecipitation
Immunoblotting
Proteins
Calcium
Kidney
Cell Line

Keywords

  • Receptor
  • Renal proximal tubular epithelial cells
  • Renin-angiotensin system
  • Signaling
  • Tyrosine kinase

ASJC Scopus subject areas

  • Physiology
  • Urology

Cite this

Angiotensin IV induces tyrosine phosphorylation of focal adhesion kinase and paxillin in proximal tubule cells. / Chen, Jiankang; Zimpelmann, Joe; Harris, Raymond C.; Burns, Kevin D.

In: American Journal of Physiology - Renal Physiology, Vol. 280, No. 6 49-6, 04.07.2001.

Research output: Contribution to journalArticle

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N2 - 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 (AT1) and type 2 (AT2) receptors and designated type 4 (AT4) receptors. We determined signaling pathways for ANG IV in a proximal tubular cell line, LLC-PK1/Cl4. In these cells, we found no specific binding of [125I]-ANG II. In contrast, ANG IV dose dependently competed for [125I]-labeled ANG IV binding, with no displacement by either ANG II, the AT1 receptor antagonist losartan, or the AT2 antagonist PD-123319. Saturation binding indicated the presence of AT4 receptors of high affinity [dissociation constant (Kd) = 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-PK1/Cl4 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.

AB - 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 (AT1) and type 2 (AT2) receptors and designated type 4 (AT4) receptors. We determined signaling pathways for ANG IV in a proximal tubular cell line, LLC-PK1/Cl4. In these cells, we found no specific binding of [125I]-ANG II. In contrast, ANG IV dose dependently competed for [125I]-labeled ANG IV binding, with no displacement by either ANG II, the AT1 receptor antagonist losartan, or the AT2 antagonist PD-123319. Saturation binding indicated the presence of AT4 receptors of high affinity [dissociation constant (Kd) = 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-PK1/Cl4 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.

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