Normal Hepatic Cell Surface Localization of the High Density Lipoprotein Receptor, Scavenger Receptor Class B, Type I, Depends on All Four PDZ Domains of PDZK1
ABSTRACT PDZK1 is a four PDZ domain-containing scaffold protein that binds to scavenger receptor class B, type I (SR-BI), the high density lipoprotein receptor, by its first PDZ domain (PDZ1). PDZK1 knock-out mice exhibit a >95% decrease in hepatic SR-BI protein and consequently an approximately 70% increase in plasma cholesterol in abnormally large high density lipoprotein particles. These defects are corrected by hepatic overexpression of full-length PDZK1 but not the PDZ1 domain alone, which partially restores SR-BI protein abundance but not cell surface expression or function. We have generated PDZK1 knock-out mice with hepatic expression of four PDZK1 transgenes encoding proteins with nested C-terminal truncations: pTEM, which lacks the three C-terminal residues (putative PDZ-binding motif), and PDZ1.2, PDZ1.2.3, or PDZ18.104.22.168, which contain only the first two, three, or four N-terminal PDZ domains, respectively, but not the remaining C-terminal sequences. Hepatic overexpression of pTEM restored normal hepatic SR-BI abundance, localization, and function. Hepatic overexpression of PDZ1.2 or PDZ1.2.3 partially restored SR-BI abundance ( approximately 12 or approximately 30% of wild type, respectively) but did not (PDZ1.2) or only slightly (PDZ1.2.3) restored hepatic SR-BI cell surface localization and function. Hepatic overexpression of PDZ22.214.171.124 completely restored SR-BI protein abundance, cell surface expression, and function (normalization of plasma cholesterol levels). Thus, all four PDZ domains in PDZK1, but not PDZ1-3 alone, are sufficient for its normal control of the abundance, localization, and therefore function of hepatic SR-BI, whereas the residues C-terminal to the PDZ4 domain, including the C-terminal putative PDZ-binding domain, are not required.
- SourceAvailable from: Ling Zhu
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- "The apparent half life of OATP1A2 immunoreactive protein in HEK 293 cells was ∼24 h under these conditions and was prolonged on co-expression of PDZK1 and NHERF1, a finding consistent with enhanced stability; OATP1A2 mRNA expression was unchanged. Interestingly, and consistent with the present findings regarding OATP1A2, PDZK1 has also been shown to enhance the stability of the scavenger receptor class B type I protein in mouse hepatocytes . "
ABSTRACT: The human organic anion transporting polypeptide 1A2 (OATP1A2) is an important membrane protein that mediates the cellular influx of various substances including drugs. Previous studies have shown that PDZ-domain containing proteins, especially PDZK1 and NHERF1, regulate the function of related membrane transporters in other mammalian species. This study investigated the role of PDZK1 and NHERF1 in the regulation of OATP1A2 in an in vitro cell model. Transporter function and protein expression were assessed in OATP1A2-transfected HEK-293 cells that co-expressed PDZK1 or NHERF1. Substrate (estrone-3-sulfate) uptake by OATP1A2 was significantly increased to ∼1.6- (PDZK1) and ∼1.8- (NHERF1) fold of control; this was dependent on the putative PDZ-binding domain within the C-terminus of OATP1A2. The functional increase of OATP1A2 following PDZK1 or NHERF1 over-expression was associated with increased transporter expression at the plasma membrane and in the whole cell, and was reflected by an increase in the apparent maximal velocity of estrone-3-sulfate uptake (Vmax: 138.9±4.1 (PDZK1) and 181.4±16.7 (NHERF1) versus 55.5±3.2 pmol*(µg*4 min)-1 in control; P<0.01). Co-immunoprecipitation analysis indicated that the regulatory actions of PDZK1 and NHERF1 were mediated by direct interaction with OATP1A2 protein. In further experiments PDZK1 and NHERF1 modulated OATP1A2 expression by decreasing its internalization in a clathrin-dependent (but caveolin-independent) manner. Additionally, PDZK1 and NHERF1 enhanced the stability of OATP1A2 protein in HEK-293 cells. The present findings indicated that PDZK1 and NHERF1 regulate the transport function of OATP1A2 by modulating protein internalization via a clathrin-dependent pathway and by enhancing protein stability.PLoS ONE 04/2014; 9(4):e94712. DOI:10.1371/journal.pone.0094712 · 3.23 Impact Factor
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- "In the liver, the four PDZ-domain containing protein PDZK1 binds to SR-BI’s C-terminus (mouse sequence: EAKL) and is responsible for the post-transcriptional control of SR-BI’s location and stability –. In PDZK1 KO mice, there is an ∼95% reduction in hepatic SR-BI protein expression, but no reduction in steroidogenic tissues, macrophages, or lung-derived endothelial cells , . "
ABSTRACT: PDZK1 is a four PDZ-domain containing cytoplasmic protein that binds to a variety of membrane proteins via their C-termini and can influence the abundance, localization and/or function of its target proteins. One of these targets in hepatocytes in vivo is the HDL receptor SR-BI. Normal hepatic expression of SR-BI protein requires PDZK1 - <5% of normal hepatic SR-BI is seen in the livers of PDZK1 knockout mice. Progress has been made in identifying features of PDZK1 required to control hepatic SR-BI in vivo using hepatic expression of wild-type and mutant forms of PDZK1 in wild-type and PDZK1 KO transgenic mice. Such in vivo studies are time consuming and expensive, and cannot readily be used to explore many features of the underlying molecular and cellular mechanisms. Here we have explored the potential to use either primary rodent hepatocytes in culture using 2D collagen gels with newly developed optimized conditions or PDZK1/SR-BI co-transfected cultured cell lines (COS, HEK293) for such studies. SR-BI and PDZK1 protein and mRNA expression levels fell rapidly in primary hepatocyte cultures, indicating this system does not adequately mimic hepatocytes in vivo for analysis of the PDZK1 dependence of SR-BI. Although PDZK1 did alter SR-BI protein expression in the cell lines, its influence was independent of SR-BI's C-terminus, and thus is not likely to occur via the same mechanism as that which occurs in hepatocytes in vivo. Caution must be exercised in using primary hepatocytes or cultured cell lines when studying the mechanism underlying the regulation of hepatic SR-BI by PDZK1. It may be possible to use SR-BI and PDZK1 expression as sensitive markers for the in vivo-like state of hepatocytes to further improve primary hepatocyte cell culture conditions.PLoS ONE 07/2013; 8(7):e69725. DOI:10.1371/journal.pone.0069725 · 3.23 Impact Factor
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- "To develop targeted delivery approaches for siRNA   , several tumor cell receptors have been examined. The SR-B1 receptor is expressed mainly in the liver and, to a lesser extent, in the adrenal glands or normal ovarian tissues     . However, SR-B1 expression in malignant cells is quite prominent. "
ABSTRACT: RNA interference holds tremendous potential as a therapeutic approach, especially in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA). To maintain a high level of growth, tumor cells scavenge high-density lipoprotein (HDL) particles by overexpressing its receptor: scavenger receptor type B1 (SR-B1). In this study, we exploited this cellular characteristic to achieve efficient siRNA delivery and established a novel formulation of siRNA by incorporating it into reconstituted HDL (rHDL) nanoparticles. Here, we demonstrate that rHDL nanoparticles facilitate highly efficient systemic delivery of siRNA in vivo, mediated by the SR-B1. Moreover, in therapeutic proof-of-concept studies, these nanoparticles were effective in silencing the expression of two proteins that are key to cancer growth and metastasis (signal transducer and activator of transcription 3 and focal adhesion kinase) in orthotopic mouse models of ovarian and colorectal cancer. These data indicate that an rHDL nanoparticle is a novel and highly efficient siRNA carrier, and therefore, this novel technology could serve as the foundation for new cancer therapeutic approaches.Neoplasia (New York, N.Y.) 04/2011; 13(4):309-19. DOI:10.1593/neo.101372 · 5.40 Impact Factor