Dominant Negative Mutants of Guanylyl Cyclase: Probes for Global Functions and Intramolecular Mechanisms

Dominant negative mutants are unique tools to define functions of a protein, not only within complex cellular and organismal contexts, but also mechanistically within a protein. Guanylyl cyclases are amenable to studies with dominant negative mutants, with their own sets of opportunities for insight and pitfalls to overcome. Membrane and soluble forms of guanylyl cyclase represent self-contained signal transduction modules that recognize, transduce, and amplify an external signal to give a carefully controlled response. Beginning with recognition of peptide hormones versus nitric oxide, membrane and soluble guanylyl cyclases are considerably different, except that their catalytic domains are closely related. Studies on these catalytic domains and their counterparts in adenylyl cyclases have raised an integral question of whether one or two domains form a catalytic site, which remains unresolved. Regardless of which model is correct, guanylyl cyclases appear to require an oligomeric state to function properly. The inferred relationship between protein–protein interaction and function is the basis for developing dominant negative mutants, which can be designed without prior structural information. Soluble guanylyl cyclases exist in a heterodimeric state, whereas membrane guanylyl cyclases are homodimeric, or possibly higher-order oligomers. These properties dictate that dominant negative mutants of membrane and soluble guanylyl cyclases be approached in fundamentally different ways, with regard to their design, their functional consequences, and their limitations. Using dominant negative mutants as specific inhibitors in complex systems, such as transgenic animals, represents a significant advance, and continuing improvements are just an inkling of the extraordinary potential of this approach. For example, the function of a protein can be obscured because it is expressed in multiple cell types; by restricting its pattern of expression, a cell-specific promoter, coupled to a dominant negative mutant, can pinpoint this function. As more sophisticated methods are developed, dominant negative mutants will provide additional opportunities to unveil new regulatory mechanisms, new signaling pathways, or even new therapeutic approaches.