EXPRESSION OF PLACENTAL GROWTH HORMONE RELATED PROTEINS

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The overall goal of sabbatical training will be to synthesize recombinant rat placental growth hormone/prolactin related protein (GHRPs) and to characterize their binding and biological activities toward different forms of receptors. My laboratory has recently identified a novel family of GHRPs in rat placenta and is funded by NIH to study the role of GHRPs in feto-placental growth. The goal of the sabbatical is to synthesize individual proteins in the GHRP family, evaluate their respective ability to bind and activate different forms of receptors, and to establish a basis for predicting potential growth-promoting effects in pregnant rats. The first specific aim is to generate cell lines which stably express cDNAs for the rat placental GHRPs and the Prolactin-Like-Proteins B and C. Culture media containing recombinant proteins will be evaluated for binding activity to native growth hormone and prolactin receptors, as well as to cloned receptors which have been stably expressed in Dr. Kelly's laboratory. The first term of the sabbatical will be from Sep 1992-Feb 1993. My laboratory at the University of Florida will subsequently purify the recombinant proteins synthesized by the transfected cell lines during the period of Mar-Aug 1992. The second specific aim is to characterize the purified recombinant proteins for binding and biological activities using cloned long and short forms of receptors expressed in CHO cells. Functionality will be evaluated using cotransfection of different PRL receptor cDNAs with a Beta-lactoglobin promoter-chloramphenicol acetyltransfersase (CAT) receptor gene construct. This work will be carried out during the second term of the sabbatical in France from Sep-Nov 1993. My laboratory in Florida will subsequently begin to characterize the purified recombinant proteins for growth-promoting effects in pregnant rats during the period of Jan-Aug 1994. During the third term of the sabbatical, from Sep-Nov 1994, the third specific aim will be to synthesize mutated forms of recombinant placental proteins using site-directed mutagenesis. The binding and biological activities of the mutated proteins will be evaluated toward cloned receptors in Dr. Kelly's laboratory. The mutated proteins will subsequently be purified and evaluated for growth-promoting activity in several animal models of low birth weight at the University of Florida.