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 alpha and a beta subunit Two heterodimeric enzymes are known to be important for NO-signalling in humans: alpha(1)/beta(1) and alpha(2)/beta(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 alpha(1)/beta(1) form more effectively. Recent evidence suggests that homodimeric complexes of alpha and beta subunits exist in vivo and that these non-heterodimerizing subunits have a separate function from cGMP signaling To isolate the effect of the alpha(1)/beta(1) or alpha(2)/beta(1) heterodimeric enzyme in overexpression experiments from potential effects of non-heterodimerizing alpha(1), beta(1) or alpha(2) subunits, we cloned constructs that guarantee a 1 1 stochiometry between alpha and beta subunits and rule out the presence of homodimers The carboxy-terminus of the beta(1) subunit was directly fused to the amino-terminus of either the alpha(1) or alpha(2) subunit The two different conjoined NOsGCs faithfully reproduced the biochemical and pharmacological properties of the alpha(1)/beta(1) and alpha(2)/beta(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 alpha or beta 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 (C) 2010 Elsevier Inc. All rights reserved