Abstract
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.
Highlights
Cytoplasmic adaptor proteins that bind directly to cell surface receptors or to receptor-associated proteins play crucial roles in regulating various biological processes including signal transduction, adhesion, membrane trafficking, and cellular transport [1]
PDZK1, a four PDZ domain-containing protein whose expression is increased in a significant number of human kidney, colon, lung, and breast carcinomas, interacts with a number of membrane-associated transporter proteins, including cMOAT/ MRP2 [3], the multidrug resistance-associated protein, the type IIa Naϩ/Pi cotransporter [4], the chloride channel ClC-3B [5], the cystic fibrosis transmembrane conductance regulator [5, 6], and the high density lipoprotein (HDL)1 receptor called scavenger receptor class B type I (SR-BI) [7, 8]
The loss of hepatic SR-BI expression in these mice is the likely cause of the elevation in total plasma cholesterol levels [14] and the presence of abnormally large HDL particles, as in the case for SR-BI knock-out mice [15], wild-type mice in which hepatic SR-BI protein expression is abolished by treatment with the peroxisome proliferator-activated receptor-␣ agonist ciprofibrate [16], and mice in which hepatic SR-BI and PDZK1 protein expression was dramatically suppressed by transgenic hepatic overexpression of the small PDZK1-interacting protein MAP17 [3, 9, 17, 18]
Summary
Animals—PDZK1 knock-out and wild-type mice (129SvEv background) were maintained on a normal chow diet [14] and were ϳ6 –12 weeks old at the time of the experiments. Immunoperoxidase studies were performed on 5-m fixed-frozen tissue sections using primary antibodies against SR-BI. The sections were incubated with a biotinylated anti-rabbit IgG using a 1/200 dilution (Vector, Burlingame, CA) and subsequently treated with the Vectastain ABC reagents (Vector) and diaminobenzidine (Research Genetics, Inc., Huntsville, AL), according to the manufacturer’s protocol. RNA Isolation—Tissues were surgically removed from PDZK1 knock-out and wild-type mice, frozen on dry ice, and stored at Ϫ80 °C until use. Plasma was fractionated by fast protein liquid chromatography, and the cholesterol and apolipoprotein compositions of the fractions were determined by enzymatic kit and immunoblotting, respectively [15]. Statistical Analysis—Data are shown as the means Ϯ S.E. Statistically significant differences were determined by pairwise comparisons of each value from knock-out mice with wild-type controls by using unpaired Student’s t test. Significant differences were determined by pairwise comparisons of each value from knock-out mice with wild-type controls by using unpaired Student’s t test. p values Ͻ 0.05 were considered to be statistically significant for differences between experimental groups
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