Interaction between the 90-kDa Heat Shock Protein and Soluble Guanylyl Cyclase: Physiological Significance and Mapping of the Domains Mediating Binding

• Published in: Molecular pharmacology 2005-10, Vol.68 (4), p.1133-1141
• Main Authors: Papapetropoulos, Andreas, Zhou, Zongmin, Gerassimou, Christina, Yetik, Gunay, Venema, Richard C, Roussos, Charis, Sessa, William C, Catravas, John D
• Format: Article
• Language: English
• Subjects: Animals; Blotting, Western; Cattle; Dimerization; Guanylate Cyclase; HSP90 Heat-Shock Proteins - antagonists & inhibitors; HSP90 Heat-Shock Proteins - chemistry; HSP90 Heat-Shock Proteins - metabolism; Immunoenzyme Techniques; Immunoprecipitation; Muscle, Smooth - metabolism; Nitric Oxide - metabolism; Protein Binding; Rats; Receptors, Cytoplasmic and Nuclear - chemistry; Receptors, Cytoplasmic and Nuclear - genetics; Receptors, Cytoplasmic and Nuclear - metabolism; Signal Transduction; Soluble Guanylyl Cyclase
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.105.012682

The 90-kDa heat shock protein (hsp90) regulates the stability and function of many client proteins, including members of the NO-cGMP signaling pathway. Soluble guanylyl cyclase (sGC), an NO receptor, was recently reported to be an hsp90-interacting partner. In the present study, we show that hsp90 binds to both subunits of the most common sGC form (α 1 β 1 ) when these are expressed individually but only interacts with β 1 in the heterodimeric form of the enzyme. Characterization of the region of hsp90 required to bind each subunit in immunoprecipitation experiments revealed that residues 310 to 456 of hsp90 interact with the sGC subunits. The region of β 1 responsible for binding to hsp90β was mapped using in vitro binding assays and immunoprecipitation experiments and was found to lie in the regulatory domain. The physiological importance of the hsp90/sGC interaction was investigated by treating rat smooth muscle cells with the hsp90 inhibitors radicicol and geldanamycin (GA) and determining both sGC activity and protein levels. Long-term (24 or 48 h) inhibition of hsp90 resulted in a strong decrease of both α 1 and β 1 protein levels and sGC activity. Moreover, incubation of smooth muscle cells with the proteasome inhibitor N -benzoyloxycarbonyl ( Z )-Leu-Leu-leucinal (MG132) blocked the GA-induced down-regulation of sGC. We conclude that the N-terminal region of the β 1 subunit mediates binding of the heterodimeric form of sGC to hsp90 and that this interaction involves the M domain of hsp90. Hsp90 binding to sGC regulates the pool of active enzymes by affecting the protein levels of the two subunits.