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.
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.
Haase
Nadine
N
University of Braunschweig - Institute of Technology, Department of Pharmacology, Toxicology and Clinical Pharmacy, Mendelssohnstrasse 1, 38106 Braunschweig, Germany.
Haase
Tobias
T
Kraehling
Jan Robert
JR
Behrends
Soenke
S
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
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