Membrane-bound and soluble proteins that bind growth hormone (GH) with high affinity are present in many species. Our studies on the hepatic GH receptor (GHR) and serum GH binding protein (GHBP) of the mouse suggest these proteins are generated by alternative splicing of a single GHR/GHBP gene to yield mRNAs encoding either membrane-bound GHR or soluble GHBP, but this hypothesis has not been proven. The primary goal of this project is to characterize the mechanism by which GHR and GHBP are generated in the mouse. Genomic blots will be analyzed to determine if there is a single GHR/GHBP gene or individual genes for each protein. Gene structure will be examined by genomic cloning, restriction mapping and sequence analysis. A second goal of this project is to develop quantitative assays for GHBP and mouse GH (mGH) and to determine gestational profiles of GHBP and mGH in maternal serum. Preliminary data indicate that GHBP interferes with measurement of mGH by existing radioimmunassays (RIAs). An RIA to accurately measure total mGH will be developed either by using a pre-assay extraction method to remove GHBP or by generating an anti-peptide antiserum that binds both free mGH and mGH bound to GHBP. Maternal serum will be fractionated to separate GHBP-bound mGH from free mGH, and the mGH concentration of both fractions will be measured to determine the fraction of mGH bound to mGHBP throughout pregnancy. To develop an RIA for GHBP, recombinant GHBP will be expressed in Chinese hamster ovary cells and purified. The recombinant GHBP will be used to develop an RIA. None of the GHRs that have been cloned has been shown to elicit a biological response. The third goal of this project is to develop a system for examining the biological effect of mGH binding to GHR. Effects of mGH on glucose uptake and oxidation will be examined in 3T3-F442A adipocytes transfected with GHR cDNA. Since this analysis may be complicated by endogenous GHR, an alternative will be to use cells transfected with a chimeric receptor composed of the extracellular domain of the mouse prolactin receptor and the transmembrane and intracellular domains of mouse GHR. Glucose uptake and oxidation in response to mouse prolactin binding to this chimeric receptor will be examined. These systems may prove very useful for future studies of signal transduction by GHR. The function of GH and the regulation of GH concentrations in maternal serum during pregnancy is not well understood. The studies in this project win provide important basic information about membrane-bound receptors for GH, which mediate GH action at the cellular level, and about soluble GH binding protein, which probably plays an important role in determining the effective concentration of GH in maternal serum.
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