This research proposal is directed toward an understanding of the structure/function relationships of multifunctional proteins that participate in major regulatory systems. Details of the organization and specific binding properties of structural and functional domains of selected proteins will be examined by methods of sequence analysis, limited proteolysis and recombination of subfragments constituting functional domains. Evidence will be sought in relation to the thesis that various cyclic nucleotide phosphodiesterases that respond to different regulatory signals belong to one protein family of homologous but chimeric character, and that this family bears a mechanistic, if not evolutionary, relationship to an analogous family of protein kinases. Domain isolation and recombination experiments will be designed to explore the mechanisms of signal transduction among representative members of these two super-gene families. It is expected that this study will lead to a more comprehensive understanding of the diverse mechanisms of regulation of enzymes and will direct attention to featrues common to regulated enzymes, whether controlled by zymogen activation, allostery or covalent modification. The proposal also describes plans to examine the multi-domain organization of profilaggrin, a protein that requires multiple posttranslational modification events (phosphorylation/ dephosphorylation and proteolysis) for its maturation and physiological function. Details of the unusual repetitive domain organization of profilaggrin will be determined. The protease(s) involved in its posttranslational processing will be identified. The chimeric domain organization of these various proteins will be compared and contrasted in efforts to develop a more comprehensive theory of the domain arrangements within multifunctional proteins.
