IDENTIFICATION OF PREF 1 INTERACTING PROTEIN

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The long-term objectives of this research are: (1) To explore the regulatory function of pref-1 in both adipocyte differentiation and embryogenesis. (2) To pursue the molecular mechanism of preadipocyte differentiation and its connection with human disease. The specific aims of this proposal are (1) to identify the pref-1 interacting protein(s); (2) to done the cDNA encoding the interacting protein(s). The primary efforts will be focused on protein(s) that interacts with the extracellular domain (ECD) of pref-1. The protein(s) will be identified biochemically by its affinity binding to purified pref-1 fusion proteins and/or endogenous expressed pref-1 protein. Three conventional methods, the GST-pulldown assay, the crosslinking assay, and the co- immunoprecipitation assay will be applied. The open reading frame encoding the pref-1 interacting protein(s) will be doned by one of the following methods: (1) using a DNA probe derived from purified interacting protein or radiolabeled GST-pref-1(ECD) to probe a cDNA expression library made from the 3T3-L1 cells; (2) yeast two-hybrid screening; 3). panning.