The long-term goal of this research is to elucidate novel mechanisms that regulate the exchange of information between cells. Of specific interest here are the biological functions for transmembrane ligands of receptor tyrosine kinases. This proposal focuses on the neuregulins, a family of multipotent cell-to-cell signaling molecules. The neuregulins and their receptors, which are receptor tyrosinekinases, play essential roles in developmental decision-making within the nervous system and heart, and they are also implicated in the pathogenesis of breast cancer. Many of the neuregulins are synthesized as transmembrane proteins. Although it is the extracellular domain of transmembrane neuregulins that activates their cognate tyrosinekinase receptors, the cytoplasmic domain sequence is more highly conserved (87% identical between humans and chickens). This stringent conservation of sequence strongly suggests involvement of the cytoplasmic domain in functions critical to the biological actions of neuregulins. Surprisingly, the neuregulin cytoplasmic domain has been largely ignored in prior studies, so its function are currently unknown. Furthermore, little is known regarding cytoplasmic domain functions for other transmembrane ligands of receptor tyrosine kinases. Two non-receptor protein kinases that interact with the cytoplasmic domain of the neuregulins have now been identified by yeast two- hybrid screening. These new results support the idea that the neuregulin cytoplasmic domain functions in regulatory pathways central to neuregulin biology. The physical and functional interactions of the neuregulins with these identified kinases will be determined in mammalian cells to confirm this hypothesized role for the neuregulin cytoplasmic domain. Co-immunoprecipitation, immunocytochemistry, and phosphorylation assays in cultured cells will be used in these first studies of neuregulin cytoplasmic domain interactions with kinases. Immunohistochemistry will be used to determine whether neuregulins and the interacting kinases are co- expressed in developing and adult motor and sensory neurons. To determine if neuregulins themselves function as receptors, experiments will test whether events affecting the neuregulin extracellular domain regulate the interactions of neuregulin and the identified kinases. To identify additional proteins mediating cytoplasmic domain function, further two-hybrid screening will be conducted. The proposed studies are expected to produce new insights into molecular mechanisms regulating developmental and pathological processes in the nervous system, heart, and breast.
