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J. Biol. Chem., Vol. 280, Issue 3, 2186-2196, January 21, 2005

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Characterization of a Novel Type of Endogenous Activator of Soluble Guanylyl Cyclase^*

Nataliya Balashova, Fu-Jung Chang, Maria Lamothe, Qian Sun, and Annie Beuve

From the Department of Pharmacology and Physiology, New Jersey Medical School, UMDNJ, Newark, New Jersey 07103

Nitric oxide (NO) remains the only firmly established endogenous modulator of soluble guanylyl cyclase (sGC) activity, but physiological, structural, and biochemical evidence now suggests that in vivo regulation of sGC involves direct interaction with other factors. We searched for such endogenous modulators in human umbilical vein endothelial cells and COS-7 cells. The cytosolic fraction of both cell types stimulated the activity of semipurified sGC severalfold in the absence or presence of a saturating concentration of NO. The cytosolic factor was sensitive to proteinase K and destroyed by boiling, suggesting that it contains a protein component. Size exclusion chromatography revealed peaks of activity between 40 and 70 kDa. The sGC-activating effect was further purified by ion exchange chromatography. In the presence of the benzylindazole YC-1 or NO, the partially purified factor synergistically activated sGC, suggesting that this factor had a mode of activation different from that of YC-1 or NO. Four candidate activators were identified from the final purification step by matrix-assisted laser desorption ionization mass spectrometry analysis. Using an sGC affinity matrix, one of them, the molecular chaperone Hsp70, was shown to directly interact with sGC. This interaction was further confirmed by co-immunoprecipitation in lung tissues and by co-localization in smooth muscle cells. sGC and Hsp70 co-localized at the plasma membrane, supporting the idea that sGC can be translocated to the membrane. Hsp70 co-purifies with the sGC-activating effect, and immunodepletion of Hsp70 from COS-7 cytosol coincided with a marked attenuation of the sGC-activating effect, yet the effect was not rescued by the addition of pure Hsp70. Thus, Hsp70 is a novel sGC-interacting protein that is responsible for the sGC-activating effect, probably in association with other factors or after covalent modification.

Received for publication, October 12, 2004

^* This work was supported by American Heart Association Grant SDG 0130506T, the Foundation of UMDNJ, and National Institutes of Health Grant RO1-GM067640. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology and Physiology, New Jersey Medical School, UMDNJ, 185 South Orange Ave., Newark, NJ 07103. Tel.: 973-972-8838; Fax: 973-972-4554; E-mail: annie.beuve{at}umdnj.edu.

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