title: Homodimerization of soluble guanylyl cyclase subunits. Dimerization analysis using a glutathione s-transferase affinity tag.
abstract: Soluble guanylyl cyclase (sGC) is an alpha/beta-heterodimeric hemoprotein that, upon interaction with the intercellular messenger molecule NO, generates cGMP. Although the related family of particulate guanylyl cyclases (pGCs) forms active homodimeric complexes, it is not known whether homodimerization of sGC subunits occurs. We report here the expression in Sf9 cells of glutathione S-transferase-tagged recombinant human sGCalpha1 and beta1 subunits, applying a novel and rapid purification method based on GSH-Sepharose affinity chromatography. Surprisingly, in intact Sf9 cells, both homodimeric GSTalpha/alpha and GSTbeta/beta complexes were formed that were catalytically inactive. Upon coexpression of the respective complementary subunits, GSTalpha/beta or GSTbeta/alpha heterodimers were preferentially formed, whereas homodimers were still detectable. When subunits were mixed after expression, e.g. GSTbeta and beta or GSTalpha and beta, no dimerization was observed. In conclusion, our data suggest the previously unrecognized possibility of a physiological equilibrium between homo- and heterodimeric sGC complexes.
authors.affiliation: Department of Pharmacology and Toxicology, Julius-Maximilians-University, D-97078 Wuerzburg, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Zabel
authors.forename: C
authors.initials: C
authors.lastname: Häusler
authors.forename: M
authors.initials: M
authors.lastname: Weeger
authors.forename: H H
authors.initials: HH
authors.lastname: Schmidt
mesh_terms: D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D002850:Chromatography, Gel; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000378:metabolism*; D006801:Humans; D011487:Protein Conformation; D012995:Solubility; D018411:Spodoptera
chemical_list: D000345:Affinity Labels; D005982:Glutathione Transferase; D006162:Guanylate Cyclase