PDZK1 Is Required for Maintaining Hepatic Scavenger Receptor, Class B, Type I (SR-BI) Steady State Levels but Not Its Surface Localization or Function

Abstract

PDZK1 is a multi-PDZ domain-containing adaptor protein that binds to the C terminus of the high density lipoprotein receptor, scavenger receptor, class B, type I (SR-BI), and controls the posttranscriptional, tissue-specific expression of this lipoprotein receptor. In the absence of PDZK1 (PDZK1(؊/؊) mice), murine hepatic SR-BI protein levels are very low (<5% of control). As a consequence, abnormal plasma lipoprotein metabolism (ϳ1.5-1.7-fold increased total plasma cholesterol carried in both normal size and abnormally large high density lipoprotein particles) resembles, but is not as severely defective The HDL 2 receptor SR-BI plays an important role in lipoprotein-mediated lipid transport and metabolism (1). SR-BI mediates HDL binding to cells and subsequently facilitates the net transfer of cholesteryl esters from the particle core but not the protein or most of the lipid components of the outer shell of the lipoprotein, a process called selective lipid uptake (1-4). This receptor also mediates bidirectional movement of unesterified cholesterol between cells and lipoproteins (5-7). SR-BI is most highly expressed in hepatocytes, where it helps control plasma lipoprotein metabolism, and in steroidogenic cells, where it delivers lipoprotein cholesterol for storage and subsequent conversion into steroid hormones (4,8,9). Normal expression and moderate transgene-mediated overexpression of hepatic SR-BI have been shown to protect against atherosclerosis in several murine models (10 -16), and SR-BI transgene expression in the liver can prevent the female infertility seen in otherwise SR-BI null mice (SR-BI (Ϫ/Ϫ)) (12,17,18).SR-BI deficiency in SR-BI (Ϫ/Ϫ) mice causes an ϳ2-fold elevation in plasma cholesterol carried in both normal size and abnormally large HDL particles (12, 19), which exhibit an abnormally high ratio of unesterified cholesterol-to-total (unesterified plus esterified) cholesterol (14, 20) as well as a 30 -50% decrease in biliary cholesterol secretion rates and concentrations (12,21,22). These phenotypes are thought to be consequences of the reduced hepatic uptake of cholesterol from circulating HDL and can be reversed by hepatic SR-BI transgene expression (17,21,23,24).Tissue SR-BI expression can be regulated by both transcriptional and posttranscriptional mechanisms (25). In the liver, but not in steroidogenic tissues, posttranscriptional control of SR-BI protein expression depends on the presence of an adaptor protein, PDZK1 (26 -28). Cytoplasmic adaptor proteins that bind to membrane-associated proteins regulate a variety of biological processes, including signal transduction, adhesion, membrane trafficking, and cellular transport (29). They often comprise combinations of modular protein interaction domains such as Src homology (SH2, SH3), phosphotyrosinebinding (PTB), and PDZ domains that recognize short peptide or phosphopeptide motifs (e.g. PDZ domains usually bind to the C-terminal 3-4 residues of interacting proteins) (30).PDZK1 is a single chain, four-PDZ domain-containing polypeptide tha...

Overexpression of the PDZ1 Domain of PDZK1 Blocks the Activity of Hepatic Scavenger Receptor, Class B, Type I by Altering Its Abundance and Cellular Localization

“…These features are similar to, although not as severe as, those found in SR-BI KO mice (19). We have previously shown that hepatic overexpression of an SR-BI transgene in SR-BI/PDZK1 double knock-out mice leads to SR-BI expression on the cell surface and normal function (43). Thus, when SR-BI is on the cell surfaces of hepatocytes at steady state levels similar to those in wild-type hepatocytes, it can function normally without PDZK1 (43).…”

“…The rabbit polyclonal anti-mSR-BI 495-112 antipeptide antibody (prepared by Invitrogen) was generated using similar procedures and against the same peptide as a previously described antimSR-BI 495 antibody and gives the same results as the anti-mSR-BI 495 antibody (3,43). The rabbit polyclonal antibody was prepared against a 30-mer amino-terminal peptide from the murine PDZK1 protein sequence, coupled to keyhole limpet hemocyanin (Genemed Synthesis, South San Francisco, CA), and affinity-purified.…”

“…We have previously shown that hepatic overexpression of an SR-BI transgene in SR-BI/PDZK1 double knock-out mice leads to SR-BI expression on the cell surface and normal function (43). Thus, when SR-BI is on the cell surfaces of hepatocytes at steady state levels similar to those in wild-type hepatocytes, it can function normally without PDZK1 (43). The mechanism by which PDZK1 regulates the abundance and/or localization of hepatic SR-BI in nontransgenic animals remains unclear.…”

The Scavenger Receptor Class B Type I Adaptor Protein PDZK1 Maintains Endothelial Monolayer Integrity

“…In hepatocytes, PDZK1 modulates the steady-state levels of SR-BI 19,30 and potentially similar mechanisms in vascular cells were evaluated. Whereas SR-BI abundance was markedly lower in livers of PDZK1 Ϫ/Ϫ versus PDZK1 ϩ/ϩ mice, levels in whole aorta were unchanged, suggesting that the mechanisms regulating SR-BI expression are different in these tissues.…”

In Vitro and in Vivo Analysis of the Binding of the C Terminus of the HDL Receptor Scavenger Receptor Class B, Type I (SR-BI), to the PDZ1 Domain of Its Adaptor Protein PDZK1

“…As described previously (9,26,27,46), absence of PDKZ1 expression in nontransgenic PDZK1 KO mice resulted in a dramatic loss of hepatic SR-BI protein (Figs. 6A, 7A, and 8B), increased plasma total cholesterol (black bars in Figs.…”