Targeted Disruption of the PDZK1 Gene in Mice Causes Tissue-specific Depletion of the High Density Lipoprotein Receptor Scavenger Receptor Class B Type I and Altered Lipoprotein Metabolism

Pontifical Catholic University of Chile, CiudadSantiago, Santiago Metropolitan, Chile
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2004; 278(52):52820-5. DOI: 10.1074/jbc.M310482200
Source: PubMed


PDZK1, a multi-PDZ domain containing adaptor protein, interacts with various membrane proteins, including the high density lipoprotein (HDL) receptor scavenger receptor class B type I (SR-BI). Here we show that PDZK1 controls in a tissue-specific and post-transcriptional fashion the expression of SR-BI in vivo. SR-BI protein expression in PDZK1 knock-out (KO) mice was reduced by 95% in the liver, 50% in the proximal intestine, and not affected in steroidogenic organs (adrenal, ovary, and testis). Thus, PDZK1 joins a growing list of adaptors that control tissue-specific activity of cell surface receptors. Hepatic expression of SR-BII, a minor splice variant with an alternative C-terminal cytoplasmic domain, was not affected in PDZK1 KO mice, suggesting that binding of PDZK1 to SR-BI is required for controlling hepatic SR-BI expression. The loss of hepatic SR-BI was the likely cause of the elevation in plasma total and HDL cholesterol and the increase in HDL particle size in PDZK1 KO mice, phenotypes similar to those observed in SR-BI KO mice. PDZK1 KO mice differed from SR-BI KO mice in that the ratio of unesterified to total plasma cholesterol was normal, females were fertile, and cholesteryl ester stores in steroidogenic organs were essentially unaffected. These differences may be due to nearly normal extrahepatic expression of SR-BI in PDZK1 KO mice. The PDZK1-dependent regulation of hepatic SR-BI and, thus, lipoprotein metabolism supports the proposal that this adaptor may represent a new target for therapeutic intervention in cardiovascular disease.

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    • "This could be important for studies of PDZK1 function. PDZK1 is a 70-kDa scaffolding protein that can assemble protein complexes, and plays an important role in regulating expression of the high-density lipoprotein scavenger receptor class B type 1 (Silver, 2002;Kocher et al., 2003) as well as cell surface expression of Oatp1a1-in the liver (Wang et al., 2005). Our studies indicate that PDZK1 is not required for binding of Oatp1a1-containing vesicles to microtubules (Fig. 1B), but that it regulates the direction in which these vesicles move (Fig. 1C). "
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    ABSTRACT: Previous studies identified a family of organic anion transport proteins (OATPs), many of which have C-terminal PDZ binding consensus sequences. In particular, the C-terminal four amino acids of oatp1a1, a transporter on rat and mouse hepatocytes, comprise a consensus binding site for PDZK1. In PDZK1 knockout mice and in transfected cells where PDZK1 expression was knocked down, oatp1a1 accumulates in intracellular vesicles. The present study tests the hypothesis that oatp1a1 traffics to and from the cell surface in vesicles along microtubules and that PDZK1 guides recruitment of specific motors to these vesicles. Oatp1a1-containing vesicles were prepared from wild type and PDZK1 knockout mice. As seen by immunofluorescence, kinesin-1, a microtubule plus-end directed motor, was largely associated with vesicles from wild type mouse liver, while dynein, a minus-end directed motor, was largely associated with vesicles from PDZK1 knockout mouse liver. Quantification of motility on directionally marked microtubules following addition of 50 μM ATP showed that wild type vesicles moved equally towards the plus and minus ends while PDZK1 knockout vesicles moved predominantly towards the minus end, consistent with net movement towards the cell interior. These studies provide a novel mechanism by which PDZK1 regulates intracellular trafficking of oatp1a1 by recruiting specific motors to oatp1a1-containing vesicles. In the absence of PDZK1, oatp1a1-containing vesicles can't recruit kinesin-1 and associate with dynein as a predominant minus-end directed motor. Whether this is a result of direct interaction of the oatp1a1 cytoplasmic domain with dynein or with a dynein-containing protein complex remains to be established.
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    • "The PDZK1-dependence of SR-BI abundance and activity in the transfected COS cells raised the possibility that this in vitro cultured cell system might mechanistically mimic the PDZK1-dependence of SR-BI in livers in vivo. To explore this possibility, we examined the effects of coexpression in COS cells of PDZK1 on the abundance of three SR-BI variants that do not have PDZK1 binding sites at their C-termini and thus either do not in vivo or are not expected to exhibit PDZK1 dependence [36], [45]. Two of these variants are C-terminal deletion mutants of SR-BI lacking either a single amino acid at position 509 (Δ509) or essentially the entire C-terminal cytosolic domain (residues 468 to 509, ‘ΔC-term’). "
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    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.
    Preview · Article · Jul 2013 · PLoS ONE
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    • "Little is known about SR-B1 regulation, yet, PDZK1 plays an important role in stabilizing SR-B1 protein in membranes [18] [19]. Pdzk1 −/− mice have a plasma cholesterol profile comparable to that of Sr- b1 −/− mice [20]. To study the potential involvement of SR-B1 and/or PDZK1 in the appearance of the large HDL upon LXR activation, we determined their hepatic gene and membranous protein expres- sion. "
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    ABSTRACT: Pharmacological LXR activation has anti-atherosclerotic actions in animal models. Part of these beneficial effects may be explained by accelerated reverse cholesterol transport since both plasma high density lipoprotein (HDL) cholesterol and fecal neutral sterol secretion are higher upon LXR activation. Mechanisms underlying these LXR-mediated effects have not been fully elucidated. We investigated the roles of the isoforms LXRα and LXRβ and the HDL cholesterol uptake receptor SR-B1 in modulation of cholesterol metabolism upon treatment of mice with the LXR ligand T0901317. HDL cholesterol was maximally 60% increased in a time-dependent fashion due to appearance of more and larger HDL particles. Fecal neutral sterol secretion was maximally induced after 1 week treatment. T0901317 treatment induced fecal neutral sterol secretion by ~300% in wild-type but not in Lxrα deficient mice. Surprisingly, LXR activation reduced SR-B1 protein amount in hepatic membranes, suggesting that this might contribute to elevated HDL cholesterol. However, T0901317 still elevated plasma HDL cholesterol in Sr-b1 deficient mice, suggesting that SR-B1 is not the only step involved in LXR-mediated induction of plasma HDL cholesterol. In addition, SR-B1 is not essential for LXR-induced cholesterol removal from the body. Induction of fecal neutral sterol secretion by T0901317 critically depends on LXRα but not on LXRβ. LXR activation reduces SR-B1 in hepatic membranes, probably partly contributing to elevated HDL cholesterol. SR-B1 is not required to enhance fecal neutral sterol secretion.
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