STRUCTURE AND FUNCTION OF GH RECEPTORS AND OF CLEAVED GH

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Recent evidence suggests that growth hormone receptors (GHR) in rabbit liver plasmalemma differ structurally from GHR in microsomal membranes, although the latter receptors also bind GH with high affinity. A late step in synthesis of the GHR, possibly occurring in the Golgi or plasmalemma, appears to involve a linking of subunits via a disulfide bond. Plasmalemmal GHR also may differ functionally in that a large proportion of the GH bound to these receptors, but not to microsomal receptors, has undergone a specific cleavage in the large disulfide loop. Cleaved hormone appears to be bound with an affinity higher than that for intact GH. To determine in which subcellular organelle(s) formation of the disulfide bond occurs, GHR in plasmalemma and in Golgi fractions (light, intermediate, heavy) will be examined by crosslinking the receptor to [125I] GH. The purity of various membrane preparations will be judged by enzyme assays and electron microscopy. Plasmalemmal and microsomal GHR will be purified using conventional techniques and various affinity chromatography columns, including a column employing as the coupled species a monoclonal antibody specific for the GRH. Individual subunits from purified GHR will be characterized according to size, isoelectric point, binding of GH and reactivity with the monoclonal antibody. Purified GHR and membrane fractions will be used to determine if activation of the GHR stimulates phosphorylation of membrane proteins, including autophosphorylation of the GHR. If phosphorylation can be demonstrated, then this endpoint will be used to examine the structural requirements for receptor activation. The role of the GHR in cleaving the GH molecule will be examined using plasmalemmal and purified GHR. The activity of cleaved hormone at binding to receptors and in stimulating a biological response will be compared to that of intact hormone. Despite innumerable studies, the function of the GHR and the nature of biologically active GH are poorly understood. However, recent developments have provided the tools necessary for a comprehensive study of GHR structure and function. This study will contribute valuabel information on the role of the receptor in mediating the actions of GH; thus, it should provide new insight into disorders of growth which are not readily attributable to deficiencies of GH or GH binding sites.