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.