Douglas L. Falls - US grants

Understanding the mechanisms of synaptic development will be central to understanding the effects of genetic and environmental factors (including drugs) on the fetus and young child. The development of the nerve-muscle synapse in vivo and in tissue culture is a valuable model of CNS synaptic development. A striking feature of this nerve-muscle synapse development is the formation of an aggregate of nicotinic acetylcholine receptors (AChR) in the postsynaptic membrane. In at least some species, an increase in the rate of insertion of AChRs into the membrane at the site of the nascent synapse makes an important contribution to the formation of this aggregate. A 42kD protein has now been purified (Usdin and Fischbach, 1986) from the chicken brain which, when present at a concentration on the order of 0.5ng/ml, causes an increase in the rate of AChR incorporation into the membrane of cultured chick myotubes and also in the number of AChR aggregates. This protein might be released by the spinal motor neuron at the site of nerve-muscle contact and induce synaptic specializations. The purpose of this project is to investigate the biological role of this protein and its mechanism of action. The specific aims are: 1) Purify enough of the 42kD acetylcholine receptor inducing activity (42kD ARIA) to provide amounts needed for production of antibodies, determination of amino-terminal sequence, and studies of the biological properties of this protein. 2) Raise monoclonal and polyclonal antibodies directed against this 42kD ARIA. 3) Use these antibody reagents to investigate the biological role of this protein. A key question is whether or not antibodies directed against this protein block the formation of synaptic specializations at sites of nerve-muscle contact. This will be investigated in vitro and in vivo. The regional distribution of the 42kD ARIA will be studied using immunofluorescence, and an ELISA assay will be developed to quantitate levels of the 42kD ARIA. The effect of focal application of this protein onto myotubes will also be examined. 4) Long-term goals include determining the full amino acid sequence of the 42kD ARIA and studying its affect on the rate of synthesis of AChR subunits and on subunit mRNA levels.

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.