Our long-term goal of this proposal is to elucidate the structure and function relationship of peptide hormone receptors using recently developed biochemical techniques, such as microsequencing and hybridoma technology. Our understanding of the hormone-receptor interaction (the first step of peptide hormone action) and following biological events has greatly advanced at cellular level in the last 10 years. However, our knowledge of the hormone-receptor interaction at molecular level is still limited, since it has been extremely difficult to purify biologically active receptors which are membrane proteins and are not abundant. The insulin receptor is thought to play an important role in insulin action. Abnormality in the receptor number and/or structure have been observed in some forms of diabetes. The receptor is a large glycoprotein with Mr=300,000-350,000 which is composed of two Mr=125,000(Alpha) and two Mr=90,000(Beta) subunits and is only available in very small quantities. We have recently succeeded in purifying biologically active insulin receptor from human placental membranes. Our ongoing project applies newly developed genetic engineering technology to determine amino acid sequence of the receptor. The projects proposed here will be performed simultaneously with the ongoing project from a different standpoint based on protein chemistry: 1) We will determine specific domains of the receptor and systematically investigate each domain in relation to insulin action using a variety of monoclonal antibodies against the receptor. 2) We will study the tertiary structure-function relationship in the insulin receptor molecule using enzymatic and chemical modification techniques, and also evaluate conformational changes of the receptor upon insulin binding by means of optical spectroscopy. These studies on the insulin receptor molecule would not only reveal the domain structure of the receptor in relation to its biological activities but also allows us to understand the exact mechanism of insulin-receptor interaction and the role of the receptor in insulin action, which would provide us new sights towards understanding diabetes and eventually curing the disease or preventing its complications.
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