Despite the central role of insulin in metabolic regulation, there are still tremendous gaps in our understanding of how insulin produces its effect on specific target tissues. The biological effects of insulin are mediated through binding to a specific cellular receptor and yet a definitive molecular weight and subunit structure of this protein cannot be assigned on the basis of available data. The objective of this project is to obtain purified human insulin receptor in quantities sufficient to allow physical and chemical characterization of the intract receptor and its subunits by standard biochemical techniques. Absolute molecular weights of the holoreceptor and of its subunits will be obtained. The source of receptors will consist of human placenta. The initial stages of this project will of necessity require the development of technology suitable for extraction of the insulin receptor from massive amounts of tissue. The purification schemes proposed combine standard procedures applicable to protein purification on a large scale with 2 novel approaches. One novel approach uses Triton X-114 for extraction and concentration of receptor. The other uses an insulin affinity matrix from which the receptor may be eluted under relatively mild conditions. The availability of inexpensive n-octylglucoside for this project will make use of this desirable detergent on a large scale practical for the first time. It is expected that this will constitute another major advance over previous approaches to receptor isolation. Molecular weights of the intact receptor and its subunits, prepared by reductive alkylation, will be assessed by high speed equilibrium in the analytical centrifuge. In addition, the amino acid and sugar composition of the isolated receptor will be determined to assess independently the partial specific volume. A definitive subunit stoichiometry for the insulin receptor is expected to result from these studies and this will establish the basis for future investigation of functional and structural properties of the receptor necessary for the translation of insulin binding into metabolic control in the intact cell.
