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

Abstract

The PDZ1 domain of the four PDZ domain-containing protein PDZK1 has been reported to bind the C terminus of the HDL receptor scavenger receptor class B, type I (SR-BI), and to control hepatic SR-BI expression and function. We generated wild-type (WT) and mutant murine PDZ1 domains, the mutants bearing single amino acid substitutions in their carboxylate binding loop (Lys ) using x-ray crystallography. In addition, we incorporated the K14A and Y20A substitutions into full-length PDZK1 liver-specific transgenes and expressed them in WT and PDZK1 knock-out mice. In WT mice, the transgenes did not alter endogenous hepatic SR-BI protein expression (intracellular distribution or amount) or lipoprotein metabolism (total plasma cholesterol, lipoprotein size distribution). In PDZK1 knock-out mice, as expected, the K14A mutant behaved like wild-type PDZK1 and completely corrected their hepatic SR-BI and plasma lipoprotein abnormalities. Unexpectedly, the 10 -20-fold overexpressed Y20A mutant also substantially, but not completely, corrected these abnormalities. The results suggest that there may be an additional site(s) within PDZK1 that bind(s) SR-BI and mediate(s) productive SR-BI-PDZK1 interaction previously attributed exclusively to the canonical binding of the C-terminal SR-BI to PDZ1.

Regulation of G Protein-Coupled Receptor Function by Na+/H+Exchange Regulatory Factors

“…These results contrast with others showing reduced basal but normal forskolin-induced HCO 3 Ϫ secretion ). More strikingly, NHERF3-null mice develop lipoprotein abnormalities and atherosclerosis (Kocher et al, 2008;Kocher et al, 2010). NHERF3 may also uniquely be associated with the intestinal organic aniontransporting polypeptide (Sugiura et al, 2010).…”

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

“…However, recent experiments suggested the existence of additional binding sites for SR-BI within PDZK1. A Tyr 20 3 Ala substitution in the conserved carboxylate binding loop of PDZ1 fully blocks SR-BI C terminus binding to PDZ1 in vitro (18). Unexpectedly, when expressed in the livers of otherwise PDZK1 null mice, a full-length PDZK1 transgene containing a Tyr 20 3 Ala substitution (18) can partially restore SR-BI expression and function, including partially correcting the lipoprotein abnormalities observed in PDZK1 KO mice (18).…”

“…is commonly accepted that SR-BI interacts via its cytoplasmic C terminus with the first PDZ domain of PDZK1 (PDZ1) (14,18). This interaction is important for controlling the expression, localization, and function of SR-BI (5, 15-17, 28).…”

“…We were surprised, therefore, to find that hepatic overexpression in PDZK1 KO mice of a mutant, full-length PDZK1 transgene, in which the PDZ1 domain was inactivated, corrected hepatic SR-BI protein expression levels, and partially restored to normal its cell membrane localization and plasma cholesterol levels (18). The inactivation of PDZ1, loss of binding to the C terminus SR-BI, was a consequence of a Tyr 20 3 Ala substitution in the target peptide of the PDZ1 carboxylate binding loop (CBL).…”

“…In addition, we incorporated several amino acid substitution mutations into full-length PDZK1 expression constructs and used them to generate transgenic WT and PDZK1 KO mice with liver-specific expression of the transgenes, including [Tyr 253 3 Ala (PDZ3)]Tg, and a double mutant [Tyr 20 3 Ala (PDZ1) ϩ Tyr 253 3 Ala (PDZ3)]Tg. We previously showed that a mutant PDZK1 transgene with an inactivating mutation in PDZ1 (Tyr 20 3 Ala) alone can almost completely correct the SR-BI-related defects in PDZK1 KO mice (18). Here we show that the hepatic expression of [Tyr 253 3 Ala (PDZ3)]Tg was able to correct all of the SR-BIrelated defects in PDZK1 KO mice, whereas the doubly substituted [Tyr 20 3 Ala (PDZ1) ϩ Tyr 253 3 Ala (PDZ3)]Tg was unable to correct these defects.…”