title: CCTeta, a novel soluble guanylyl cyclase-interacting protein.
abstract: Nitric oxide (NO) transduces most of its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC). Activation of sGC results in the production of cGMP from GTP. In this paper, we demonstrate a novel protein interaction between CCT (chaperonin containing t-complex polypeptide) subunit eta and the alpha1beta1 isoform of sGC. CCTeta was found to interact with the beta1 subunit of sGC via a yeast-two-hybrid screen. This interaction was then confirmed in vitro with a co-immunoprecipitation from mouse brain. The interaction between these two proteins was further supported by a co-localization of the proteins within rat brain. Using the yeast two-hybrid system, CCTeta was found to bind to the N-terminal portion of sGC. In vitro assays with purified CCTeta and Sf9 lysate expressing sGC resulted in a 30-50% inhibition of diethylamine diazeniumdiolate-NO-stimulated sGC activity. The same assays were then performed using BAY41-2272, an NO-independent allosteric sGC activator, and CCTeta had no effect on this activity. Furthermore, CCTeta had no effect on basal or sodium nitroprusside-stimulated alphabeta(Cys-105) sGC, a constitutively active mutant that only lacks the heme group. The N-terminal 94 amino acids of CCTeta seem to be critical for the mediation of this inhibition. Lastly, a 45% inhibition of sGC activity by CCTeta was seen in vivo in BE2 cells stably transfected with CCTeta and treated with sodium nitroprusside. These data suggest that CCTeta binds to sGC and, in cooperation with some other factor, inhibits its activity by modifying the binding of NO to the heme group or the subsequent conformational changes.
authors.affiliation: Department of Integrative Biology and Pharmacology and Institute of Molecular Medicine, University of Texas Medical School, Houston, Texas 77030, USA.
authors.forename: Khalid A
authors.initials: KA
authors.lastname: Hanafy
authors.forename: Emil
authors.initials: E
authors.lastname: Martin
authors.forename: Ferid
authors.initials: F
authors.lastname: Murad
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000737:chemistry* / Q000378:metabolism*; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism*; D006624:Hippocampus / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007313:Insecta; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011863:Radioimmunoassay; D051381:Rats; D014018:Tissue Distribution; D014162:Transfection; D020798:Two-Hybrid System Techniques; D000519:alpha-Galactosidase / Q000378:metabolism
chemical_list: C533313:Cct7 protein, mouse; D020033:Protein Isoforms; C533315:Tcp1 protein, mouse; D009599:Nitroprusside; D009569:Nitric Oxide; D006639:Histidine; D006160:Guanosine Triphosphate; D000519:alpha-Galactosidase; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins; D006162:Guanylate Cyclase; D006152:Cyclic GMP