Endothelin-1 mobilizes profilin-1-bound PIP2 in cardiac muscle.
Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key down-stream substrate of the endothelin signaling pathway and plays a role in regulating protein function at the membrane-cytoskeletal interface. However, the dynamic properties of distinct pools of PIP2 are poorly understood, especially for PIP2 that is bound to cytoskeletal proteins. We investigated the effects of endothelin-1 (ET-1) stimulation on protein-bound PIP2 in cardiac muscle. Isolated rat myocytes and homogenized mouse ventricles were exposed to 10 nM ET-1 for varying time periods and protein-bound PIP2 was analyzed using an anti-PIP2 antibody and Western blotting. Several cytoskeletal proteins were found to contain tightly bound PIP2, including profilin-1 (approximately 15 kDa), capZ (approximately 32 kDa), gCap39, (approximately 39 kDa) and alpha-actinin (approximately 106 kDa). Interestingly, ET-1 pretreatment reduced the amount of PIP2 bound to profilin-1 by 46% after 15 mins, followed by a recovery to near basal levels after 60 mins. ET-1 had no effect on capZ-, gCap39-, or alpha-actinin-bound PIP2 levels. To further explore the dynamics of PIP2 binding, brefeldin-A (BFA) was used to disrupt PIP2 binding to ADP-ribosylation factors and to impair receptor internalization. Pretreatment with 1 microM BFA increased the PIP2 signal on profilin-1 x 54% after 15 mins, followed by a decline to subbasal levels after 60 mins. Like ET-1, BFA had no effect on levels of PIP2 bound to capZ or to alpha-actinin. Taken together, the data indicate that profilin-1 binds PIP2 dynamically and may serve as a key regulator of the balance between cytoskeletal integrity and PIP2 availability for Ca2+/PKC signaling in the heart.
Evans
Nathan J
NJ
Department of Physiology, University of Wisconsin--Madison, 1300 University Avenue, Madison, Wisconsin 53706, USA.
Walker
Jeffery W
JW
D000818:Animals; D019332:Endothelin-1 / Q000494:pharmacology*; D006352:Heart Ventricles / Q000166:cytology; D066298:In Vitro Techniques; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D051304:Profilins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
D019332:Endothelin-1; D019269:Phosphatidylinositol 4,5-Diphosphate; D051304:Profilins