Influence of PDZK1 on lipoprotein metabolism and atherosclerosis

PDZK1 is a scaffold protein containing four PDZ protein interaction domains, which bind to the carboxy termini of a number of membrane transporter proteins, including ion channels (e.g., CFTR) and cell surface receptors. One of these, the HDL receptor, scavenger receptor class B type I (SR-BI), exhibits a striking, tissue-specific dependence on PDZK1 for its expression and activity. In PDZK1 knockout (KO) mice there is a marked reduction of SR-BI protein expression (approximately 95%) in the liver, but not in steroidogenic tissues or, as we show in this report, in bone marrow- or spleen-derived macrophages, or lung-derived endothelial cells. Because of hepatic SR-BI deficiency, PDZK1 KO mice exhibit dyslipidemia characterized by elevated plasma cholesterol carried in abnormally large HDL particles. Here, we show that inactivation of the PDZK1 gene promotes the development of aortic root atherosclerosis in apolipoprotein E (apoE) KO mice fed with a high fat/high cholesterol diet. However, unlike complete SR-BI-deficiency in SR-BI/apoE double KO mice, PDZK1 deficiency in PDZK1/apoE double knockout mice did not result in development of occlusive coronary artery disease or myocardial infarction, presumably because of their residual expression of SR-BI. These findings demonstrate that deficiency of an adaptor protein essential for normal expression of a lipoprotein receptor promotes atherosclerosis in a murine model. They also define PDZK1 as a member of the family of proteins that is instrumental in preventing cardiovascular disease by maintaining normal lipoprotein metabolism.     

Identification of the PDZ3 domain of the adaptor protein PDZK1 as a second, physiologically functional binding site for the C terminus of the high density lipoprotein receptor scavenger receptor class B type I

The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1–PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI (“target peptide”) binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr²⁰ → Ala) only partially, rather than completely, suppresses the ability of PDZK1 to control hepatic SR-BI. We used isothermal titration calorimetry to show that PDZ3, but not PDZ2 or PDZ4, can also bind the target peptide (K_d = 37.0 μm), albeit with ∼10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr²⁵³ → Ala substitution. Comparison of the 1.5-Å resolution crystal structure of PDZ3 with its bound target peptide (⁵⁰⁵QEAKL⁵⁰⁹) to that of peptide-bound PDZ1 indicated fewer target peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr²⁰ → Ala (PDZ1) + Tyr²⁵³ → Ala (PDZ3)) substitution abrogated all target peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr²⁵³ → Ala (PDZ3)) PDZK1 transgene (Tg) was able to correct all of the SR-BI-related defects in PDZK1 knock-out mice, whereas the doubly substituted [Tyr²⁰ → Ala (PDZ1) + Tyr²⁵³ → Ala (PDZ3)]Tg was unable to correct these defects. Thus, we conclude that PDZK1-mediated control of hepatic SR-BI requires direct binding of the SR-BI C terminus to either the PDZ1 or PDZ3 domains, and that binding to both domains simultaneously is not required for PDZK1 control of hepatic SR-BI.     

Loss of PDZK1 Causes Coronary Artery Occlusion and Myocardial Infarction in Paigen Diet-Fed Apolipoprotein E Deficient Mice

Background

PDZK1 is a four PDZ-domain containing protein that binds to the carboxy terminus of the HDL receptor, scavenger receptor class B type I (SR-BI), and regulates its expression, localization and function in a tissue-specific manner. PDZK1 knockout (KO) mice are characterized by a marked reduction of SR-BI protein expression (∼95%) in the liver (lesser or no reduction in other organs) with a concomitant 1.7 fold increase in plasma cholesterol. PDZK1 has been shown to be atheroprotective using the high fat/high cholesterol (‘Western’) diet-fed murine apolipoprotein E (apoE) KO model of atherosclerosis, presumably because of its role in promoting reverse cholesterol transport via SR-BI.

Principal Findings

Here, we have examined the effects of PDZK1 deficiency in apoE KO mice fed with the atherogenic ‘Paigen’ diet for three months. Relative to apoE KO, PDZK1/apoE double KO (dKO) mice showed increased plasma lipids (33% increase in total cholesterol; 49 % increase in unesterified cholesterol; and 36% increase in phospholipids) and a 26% increase in aortic root lesions. Compared to apoE KO, dKO mice exhibited substantial occlusive coronary artery disease: 375% increase in severe occlusions. Myocardial infarctions, not observed in apoE KO mice (although occasional minimal fibrosis was noted), were seen in 7 of 8 dKO mice, resulting in 12 times greater area of fibrosis in dKO cardiac muscle.

Conclusions

These results show that Paigen-diet fed PDZK1/apoE dKO mice represent a new animal model useful for studying coronary heart disease and suggest that PDZK1 may represent a valuable target for therapeutic intervention.     

Homo-oligomeric complexes of the yeast alpha-factor pheromone receptor are functional units of endocytosis

alpha-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. Receptors solubilized with the detergent n-dodecyl beta-D-maltoside were found to sediment as a single 8S species in glycerol density gradients. When the membranes from cells coexpressing two differentially tagged receptors were solubilized with detergent and subjected to immunoprecipitation, we found that the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Coprecipitation was not a consequence of incomplete detergent extraction because the abundant plasma membrane protein Pma1 did not coprecipitate with the receptors. Moreover, the receptor complexes were present prior to detergent extraction because coimmunoprecipitation was not observed when cells expressing the single tagged species were mixed prior to membrane preparation. Treatment of cultures with alpha-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind alpha-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. We conclude that alpha-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. Furthermore, we show for the first time that unoccupied receptors participate in these endocytosis-competent complexes.