Interaction between the 90-kDa heat shock protein and soluble guanylyl cyclase: physiological significance and mapping of the domains mediating binding.
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 (alpha(1)beta(1)) when these are expressed individually but only interacts with beta(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 beta(1) responsible for binding to hsp90beta 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 alpha(1) and beta(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 beta(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.
Papapetropoulos
Andreas
A
G.P. Livanos and M. Simou Laboratories, Evangelismos Hospital, Department of Critical Care and Pulmonary Services, University of Athens, Greece. apapapet@upatras.gr
Zhou
Zongmin
Z
Gerassimou
Christina
C
Yetik
Gunay
G
Venema
Richard C
RC
Roussos
Charis
C
Sessa
William C
WC
Catravas
John D
JD
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D019281:Dimerization; D006162:Guanylate Cyclase; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D009130:Muscle, Smooth / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D000071756:Soluble Guanylyl Cyclase
D018841:HSP90 Heat-Shock Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D009569:Nitric Oxide; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase