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

Abstract

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 (Tyr 20 3 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 M), albeit with ϳ10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr 253 3 Ala substitution. Comparison of the 1.5-Å resolution crystal structure of PDZ3 with its bound target peptide ( 505 QEAKL 509 ) to that of peptide-bound PDZ1 indicated fewer target peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr 20 3 Ala (PDZ1) ؉ Tyr 253 3 Ala (PDZ3)) substitution abrogated all target peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr 253 3 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 [Tyr 20 3 Ala (PDZ1) ؉ Tyr 253 3 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.The expression and function of numerous proteins, including cell surface receptors, is controlled, at least in part, by interacting cytoplasmic adaptor proteins. These adaptors can play essential roles in regulating the functions of their targets, including signal transduction (1). PDZ (PSD-95/Discs-large/ ZO-1) domains are a family of protein interaction domains (ϳ250 in the human genome) found in over 100 proteins and can interact with and regulate the functions of a number of membrane-associated proteins (2). As PDZ domain-containing proteins often contain more than one PDZ domain, they are able to serve as scaffolds that can organize multiprotein complexes and thus influence cellular function. PDZK1 is a 519-amino acid cytoplasmic protein containing four PDZ domains (PDZ1-PDZ4) involved in the regulation of several membraneassociated proteins, including ion channels and cell-surface receptors (3, 4). Indeed, PDZK1 plays a major role in regulating the expression, localization, and function of the HDL-receptor scavenger receptor class B type I (SR-BI) 2 in a tissue-specific manner (5).SR-BI is a 509-amino acid integral membrane protein with a large extracellular loop, two transmembrane domains, and very short cytoplasmic N and C termini (6). It plays a major role in the transfer of cholestero...

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)

“…Ikemoto et al (7) first showed that the C-terminal tail of SR-BI binds to the N-terminal PDZ domain (PDZ1) of PDZK1. We confirmed this high affinity (K d ϳ2.6 M) binding in vitro, and we also found lower affinity (K d ϳ37.0 M) binding of the C terminus of SR-BI to the third PDZ domain (PDZ3) of PDZK1 but no binding to PDZ2 or PDZ4 (18,19). The C terminus of SR-BI binds to the canonical carboxylate binding loops of PDZ1 and PDZ3 (18,19), the binding site used by most PDZ domains to bind their target peptides (20).…”

“…1C) can do so (23). Thus, even though PDZ4 does not bind directly to SR-BI's C terminus (19), this domain in PDZK1 performs a critical role(s) in promoting normal SR-BI protein expression and activity.…”

“…A C-terminally truncated PDZK1 containing only the first three N-terminal PDZ domains (PDZ123) is unable to restore full, normal expression and function of SR-BI in PDZK1 KO mice, whereas a truncated PDZK1 transgene containing all four PDZ domains (PDZ1234), but missing the C-terminal 58 amino acids of the protein, is fully functional (23). These findings suggested that the fourth PDZ domain (PDZ4), which does not bind directly to SR-BI's C terminus (19), performs a critical role in promoting normal SR-BI function.…”

“…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.…”

“…Analyses of a variety of such transgenic mice established that PDZK1 controls SR-BI's expression, localization, and function. For example, we introduced point mutations in PDZK1 that disrupt the binding of either the PDZ1 or PDZ3 domains to SR-BI's C terminus and showed that only one active SR-BI-binding PDZ domain is necessary for normal PDZK1-dependent regulation of hepatic SR-BI (18,19). A C-terminally truncated PDZK1 containing only the first three N-terminal PDZ domains (PDZ123) is unable to restore full, normal expression and function of SR-BI in PDZK1 KO mice, whereas a truncated PDZK1 transgene containing all four PDZ domains (PDZ1234), but missing the C-terminal 58 amino acids of the protein, is fully functional (23).…”