Studies from our laboratory have shown that the half-time for secretion of the acute phase protein, C-reactive protein (CRP), by rabbit hepatocytes decreases dramatically in conjunction with its increased synthesis during the acute phase response and that this is due to downregulation of a specific high affinity binding site for CRP which ordinarily retains this protein within the ER of normal hepatocytes. More recently, we have identified and purified two 60 kD proteins (or two different modified forms of the same protein) with different net charges, designated gp6Oa and gp6Ob, which fulfil the necessary criteria for the previously observed high and low affinity binding site for CRP. The overall goal of this proposal is to elucidate the mechanisms which regulate the activity of the CRP binding site during the acute phase response and to directly investigate its role in the regulation of CRP secretion.
Specific aims i nclude 1) Characterization of the specificity and affinity of CRP's binding to gp6O's employing biotinated CRP and immobilized gp6O in an ELISA. 2) Peptide mapping and microsequencing will be used to determine whether the two forms represent a single protein and polyclonal antibodies will be raised in guinea pigs. 3) Synthesis, stability, and potential modification of gp6O will be determined in primary hepatocyte cultures incubated with either labeled methionine, phosphorus, myristate, or sulfate employing immunoprecipitation and SDS-PAGE. 4) Subcellular localization of gp6O's will be investigated in saponin-permeabilized hepatocytes treated with anti-gp6O followed by biotinated goat anti-guinea pig Ig antibody. 5) cDNA clones for gp6O will be isolated using degenerate oligonucleotide primers in a PCR amplification of an available rabbit cDNA library. cDNA transfection studies will allow for direct investigation of gp6O, or its modification, in the retention of CRP within the ER. The serum concentration of CRP is markedly elevated in a number if inflammatory diseases and CRP itself appears to play a role in the modulation of the inflammatory response. Investigation of mechanisms which control CRP secretion will contribute to our understanding of the host response to a wide variety of inflammatory conditions, including many of the rheumatic diseases.
