Many cells, including some tumor cells, appear to secrete autocrine factors that repress their proliferation. In most cases the factors and associated signal transduction pathways are unknown. The PI has found that growing Dictyostelium cells secrete a protein called AprA for autocrine proliferation repressor. AprA is a 60 kDa protein, is part of a secreted approximately 150 kDa complex, and has some similarity to bacterial and mammalian proteins of unknown function. Compared to wild-type cells, aprA null cells proliferate faster while AprA overexpressing cells proliferate slower. Furthermore, adding immunoprecipitated AprA to cells slows their proliferation. I propose three specific aims to elucidate the mechanisms by which AprA regulates cell proliferation. First, we will determine if the approximately 150 kDa factor contains AprA alone or AprA plus additional factors, by purifying this factor and identifying its components. Second, we will determine if our candidate receptor for this factor senses and binds this factor, or is part of a different pathway that inhibits proliferation. Third, we will identify downstream components that are required for the growth inhibitory action of this factor. In preliminary screens, we identified four suppressors of the slow-proliferation AprA overexpressor phenotype;one encodes a protein with similarity to CLN2, a gene implicated in juvenile neuronal ceroid lipofuscinosis, poorly differentiated tumors, and cardiac hypertrophy. We will make nulls of suppressor genes and determine if they are part of the AprA signal transduction pathway, or are part of a different mechanism that represses proliferation. Our identification of the aprA mutant and AprA protein has opened the way for the application of molecular and genetic analysis to the mechanism by which extracellular factors can inhibit proliferation. We anticipate that these studies will assist in understanding both how normal tissue and tumor growth are regulated in multicellular organisms, and may help lead to novel ways to repress tumor cell proliferation.
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