The mechanism of insulin (INS) action is not known. The insulin receptor (IR) mediates INS binding and early events leading to the pleiotropic effects of INS. The beta-subunit of the IR is phosphorylated on tyrosine in response to INS binding in intact cells (i.e. autophosphorylation). Much data indicate that beta- subunit autophosphorylation is required for several bioeffects of INS. Autophosphorylation activates a tyrosine kinase domain in the beta-subunit with activity towards exogenous protein substrates. Several groups have identified potential endogenous protein substrates of the IR tyrosine kinase including a 160 kDA protein (pp160) in 3T3-L1 adipocytes identified in our laboratory. Tyrosine phosphorylation of pp160 is maximal within 20 sec after INS addition to intact cells and its half-maximal in the presence of 3.5 nM INS. Our long-term goal is to determine the role of pp160 in the mediation of INS action. We propose to purify pp160 to homogeneity from 3T3-L1 cells employing both classical methods for protein separation and immunoaffinity chromatographic techniques using polyclonal anti- phosphotyrosine (alpha-PTYR) and anti-pp160 antibodies. The biochemical properties of purified pp160 will be assessed, as will the effect(s) of phosphorylation on these properties. Using peptide sequence data to be obtained from purified pp160, we propose to synthesize peptides homologous to, as well as oligonucleotides encoding these sequences. We propose to clone the cDNA encoding pp160 from an established 3T3-L1 expression library using antibodies directed against either purified pp160 or synthetic peptides derived from pp160, or oligonucleotides complementary to pp160 coding sequences. Finally, we propose to inhibit pp160 synthesis in intact cells using anti-sense RNA.
This final aim may provide clear insight into the role of pp160 in mediating one or several of insulin's actions. Future goals involve using cDNA probes to the pp160 gene, anti-pp160 antibodies, and alpha-PTYR antibodies to study the effects of various long- and short-term hormonal treatments (i.e. INS, epinephrine, glucagon, growth hormone, cortisol, etc.) and effectors (i.e. phorbol esters) on pp160 expression, translation, turnover, and phosphorylation in several different cell types. Cellular resistance to INS action is a major pathogenic factor in non-insulin dependent diabetes mellitus (NIDDM). The mechanism of INS resistance is poorly understood. This proposal is directed toward an understanding of normal IR biochemistry. This knowledge can then be used to study INS resistant states such as NIDDM.
