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

Abstract

PDZK1 is a four-PDZ domain-containing scaffold protein that, via its first PDZ domain (PDZ1), binds to the C terminus of the high density lipoprotein (HDL) receptor scavenger receptor, class B, type I (SR-BI). Abolishing PDZK1 expression in PDZK1 knock-out (KO) mice leads to a post-transcriptional, tissue-specific decrease in SR-BI protein level and an increase in total plasma cholesterol carried in abnormally large HDL particles. Here we show that, although hepatic overexpression of PDZK1 restored normal SR-BI protein abundance and function in PDZK1 KO mice, hepatic overexpression of only the PDZ1 domain was not sufficient to restore normal SR-BI function. In wild-type mice, overexpression of the PDZ1 domain overcame the activity of the endogenous hepatic PDZK1, resulting in a 75% reduction in hepatic SR-BI protein levels and intracellular mislocalization of the remaining SR-BI. As a consequence, the plasma lipoproteins in PDZ1 transgenic mice resembled those in PDZK1 KO mice (hypercholesterolemia due to large HDL). These results indicate that the PDZ1 domain can control the abundance and localization, and therefore the function, of hepatic SR-BI and that structural features of PDZK1 in addition to its SR-BI-binding PDZ1 domain are required for normal hepatic SR-BI regulation.The high density lipoprotein (HDL) 3 receptor SR-BI (scavenger receptor, class B, type I) plays a key role in lipoprotein metabolism (1) by mediating the cellular uptake of cholesteryl esters from HDL and other lipoproteins into cells (2-7) via a mechanism called selective lipid uptake (3, 8 -10). SR-BI also mediates bidirectional flux of unesterified cholesterol between cells and lipoproteins (11)(12)(13)(14).Most in vivo analyses of the physiological function of SR-BI have been conducted in mice. SR-BI is highly expressed in the liver and steroidogenic tissues (adrenal gland, ovary, and testis), which exhibit the highest levels of HDL cholesterol uptake (3). Experimental manipulations of murine SR-BI expression (e.g. hepatic overexpression, homozygous null gene knock-out) can lead to changes in total plasma cholesterol levels, the ratio of plasma unesterified to total cholesterol, HDL structure, biliary cholesterol secretion, the amounts of cholesterol stored in steroidogenic tissues, and susceptibility to atherosclerosis (15-27). Homozygous null SR-BI knock-out mice exhibit abnormally elevated (ϳ2.2-fold) plasma cholesterol in large HDL particles with a unesterified cholesterol/total cholesterol ratio roughly double that of wild-type (WT) mice (19,52). This dyslipidemia is associated with female infertility and defects in the maturation and/or structure as well as reductions in the survival times of red blood cells (21,28,29,30).To date, only one intracellular protein has been shown to interact directly with and influence the function of SR-BI (31). This protein, PDZK1, regulates SR-BI expression in a tissuespecific, post-transcriptional manner (32). PDZK1 is a scaffold protein containing four PDZ protein interaction domains (Fig. 1A...

Noncanonical Role of the PDZ4 Domain of the Adaptor Protein PDZK1 in the Regulation of the Hepatic High Density Lipoprotein Receptor Scavenger Receptor Class B, Type I (SR-BI)

“…Thus, these mutated transgenes did not produce substantial dominant-negative effects when overexpressed in WT mice (an example of a dominantnegative truncated PDZK1 is described in Ref. 24).…”

“…Vectors encoding PDZK1 with Y388A, G389P, or G389A single residue substitutions or Y388G/G389P, Y388A/G389A, or Y388A/G389P double residue substitutions within PDZ4's carboxylate binding loop were produced using a previously described wild-type PDZK1/pLiv-LE6 or PDZK1/pCDNA3.1 recombinant plasmids (24) and the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. Oligonucleotide primers containing the corresponding mutations were synthesized and PAGE-purified by Integrated DNA Technologies (IDT).…”

“…A PDZK1 cDNA gene in which the second PDZ domain (PDZ2, residues 133-212) was replaced by the PDZ1 sequence (residues 7-86, resulting in two PDZ1 domains in tandem (PDZ231)) and the PDZK1 cDNA genes in which either the PDZ2 (⌬PDZ2, residues 133-212) or PDZ3 (⌬PDZ3, residues 241-319) domains were deleted were synthesized by IDT. KpnI-XhoI DNA fragments corresponding to each of these mutant PDZK1 constructs were subcloned into the pLiv-LE6 plasmid (24), which was kindly provided by Dr. John M. Taylor (Gladstone Institute of Cardiovascular Disease, University of California, San Francisco), and contains the promoter, first exon, first intron, and part of the second exon of the human apoE gene, the polyadenylation sequence, and a part of the hepatic control region of the apoE/C-I gene locus (25). The sequences of the resulting plasmids were confirmed by DNA sequencing.…”

“…Plasma and liver samples were collected and processed, and total plasma cholesterol levels and size-fractionated FPLC cholesterol profiles were obtained as described previously (24). Plasma cholesterol results presented here for transgenic mice are pooled from data collected from animals derived from the different founder transgenic animals because the results obtained were not significantly different between founders expressing the same transgene.…”

“…As a result, PDZK1 KO mice show an increase of plasma cholesterol (ϳ1.7-fold) in the form of abnormally large HDL particles that is similar to, but not as severe as, that seen in SR-BI KO mice (8). Because we have been unable to robustly reproduce the PDZK1 dependence of SR-BI in cultured cells, 4 we have analyzed the functional consequences of altering PDZK1's structure on its function using PDZK1 transgenes expressed in hepatocytes of wild-type (WT) or PDZK1 KO mice (18,19,23,24). Analyses of a variety of such transgenic mice established that PDZK1 controls SR-BI's expression, localization, and function.…”