title: Direct fusion of subunits of heterodimeric nitric oxide sensitive guanylyl cyclase leads to functional enzymes with preserved biochemical properties: evidence for isoform specific activation by ciguates.
abstract: Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme consisting of an α and a β subunit. Two heterodimeric enzymes are known to be important for NO-signalling in humans: α(1)/β(1) and α(2)/β(1). No difference had so far been detected with respect to their pharmacological properties, but as we show in the present paper the new drugs cinaciguat and ataciguat activate the α(1)/β(1) form more effectively. Recent evidence suggests that homodimeric complexes of α and β subunits exist in vivo and that these non-heterodimerizing subunits have a separate function from cGMP signaling. To isolate the effect of the α(1)/β(1) or α(2)/β(1) heterodimeric enzyme in overexpression experiments from potential effects of non-heterodimerizing α(1), β(1) or α(2) subunits, we cloned constructs that guarantee a 1:1 stochiometry between α and β subunits and rule out the presence of homodimers. The carboxy-terminus of the β(1) subunit was directly fused to the amino-terminus of either the α(1) or α(2) subunit. The two different "conjoined" NOsGCs faithfully reproduced the biochemical and pharmacological properties of the α(1)/β(1) and α(2)/β(1) heterodimeric enzymes including the differential activation by ciguat-activators. Conjoined NOsGCs can be used for isoform specific overexpression in transgenic animals and therapeutic overexpression may be an application in the future. In both cases possible side effects of homodimeric α or β subunits are avoided. Crystallization with the goal of structure determination may also be easier for conjoined NOsGCs because enzyme preparations are more homogenous and are free of "contaminating" homodimers.
authors.affiliation: University of Braunschweig - Institute of Technology, Department of Pharmacology, Toxicology and Clinical Pharmacy, Mendelssohnstrasse 1, 38106 Braunschweig, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Haase
authors.forename: Tobias
authors.initials: T
authors.lastname: Haase
authors.forename: Jan Robert
authors.initials: JR
authors.lastname: Kraehling
authors.forename: Soenke
authors.initials: S
authors.lastname: Behrends
mesh_terms: D000818:Animals; D001565:Benzoates / Q000494:pharmacology*; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D006162:Guanylate Cyclase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009569:Nitric Oxide / Q000502:physiology*; D011485:Protein Binding / Q000235:genetics; D055503:Protein Multimerization / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000378:metabolism*; D000071756:Soluble Guanylyl Cyclase; D018411:Spodoptera
chemical_list: D001565:Benzoates; C000605660:GUCY1A2 protein, human; C000605667:Gucy1a2 protein, rat; D007527:Isoenzymes; D021122:Protein Subunits; D018160:Receptors, Cytoplasmic and Nuclear; D009569:Nitric Oxide; C480588:BAY 58-2667; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase