C-reactive protein (CRP) is normally synthesized by hepatocytes at relatively low rates and is retained within the endoplasmic reticulum (ER) via interaction with two carboxylesterases (termed gp60a and gp60b), which themselves are restricted to the ER by their COOH-terminal retention signals (HIEL and HTEL).During the acute phase response, an increase in CRP synthesis is accompanied by a decrease in its ER retention as a result of a decrease in the CRP binding affinity of gp60b. Our previous data indicated that the esterase active site, the CRP binding site, and the ER retention signal are functionally distinct. In the present studies, we have identified CRP-binding peptides produced by proteolytic cleavage of gp60a. The sequence shared by two CRP-binding peptides indicated that the CRP binding site of gp60a is contained within residues 477-499. These results were confirmed by expression of cDNAs coding for gp60a and b as bacterial fusion proteins. Fusion proteins containing the complete esterase COOH terminus bound CRP, whereas those truncated at residue 477 (or the homologous site in gp60b) did not. Based on the known crystal structures of three homologous hydrolases, the putative CRP-binding site of the gp60s is located on the surface and is physically distant from the esterase active site and the COOH-terminal ER retention signal. C-reactive protein (CRP)1 is a plasma protein whose rate of synthesis by hepatocytes may increase by several hundred-fold or more during the systemic acute phase response to tissue injury (1). CRP is a homopentamer composed of 24-kDa nonglycosylated subunits (2) that assemble within the endoplasmic reticulum (ER) shortly after their synthesis (3). Under normal physiologic conditions CRP is synthesized at relatively low rates and is largely retained within the ER without being degraded: the half-time for exit of pulse-labeled CRP from the ER in hepatocytes isolated from normal rabbits was found to be in excess of 18 h (3, 4), whereas CRP was efficiently secreted with a half-time of only 75 min in cells isolated from animals stimulated in vivo to undergo the acute phase response (4). This differential retention of CRP within the cell represents a novel mechanism capable of regulating the intracellular sorting of a secretory protein.We have previously shown that retention of CRP within the ER is the result of its association with two 60-kDa glycosylated microsomal carboxylesterases (5, 6), which we have termed gp60a and gp60b (7). gp60a is an abundant ER protein with a relatively low affinity for CRP (K d ϭ 120 nM), whereas gp60b is a less abundant protein, but with a higher binding affinity for CRP (K d ϭ 1 nM) (3, 7). These esterases are restricted to the lumen of the ER by virtue of their carboxyl-terminal sequences of HIEL and HTEL (5, 6), which have been shown to be effective ER retention signals (8, 9), presumably being recognized by the KDEL retrieval pathway (10). Thus, we have proposed that the retention of CRP within the ER is mediated by its interaction with gp60a and b, w...
To evaluate the possible role of posttranscriptional mechanisms in the acute phase response, we determined the kinetics of transcription (by nuclear run-on assay) and mRNA accumulation of five human acute phase genes in Hep 3B cells incubated with conditioned medium from LPS-stimulated monocytes. Increase in mRNA accumulation was comparable to increase in transcription rate for fibrinogen-a and a-i protease inhibitor, suggesting largely transcriptional regulation. In contrast, mRNA accumulation was about 10-20-fold greater than transcriptional increase for serum amyloid A, C3, and factor B, suggesting participation of posttranscriptional mechanisms. Since finding a disparity between the magnitudes of increase in mRNA and transcription does not definitively establish involvement of posttranscriptional mechanisms, we subjected our data to modeling studies and dynamic mathematical analysis to evaluate this possibility more rigorously. In modeling studies, accumulation curves resembling those observed for these three mRNAs could be generated from the nuclear run-on results only if posttranscriptional regulation was assumed. Dynamic mathematical analysis of relative transcription rates and relative mRNA abundance also strongly supported participation of posttranscriptional mechanisms. These observations suggest that posttranscriptional regulation plays a substantial role in induction of some, but not all acute phase proteins. (J. Clin. Invest. 1995Invest. . 95:1253Invest. -1261
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