The PDZ domain protein CAL interacts with mGluR5a and modulates receptor expression

Abstract

1These authors contributed equally to this study.Abbreviations used: CAL, CFTR-associated ligand; CFTR, cystic fibrosis transmembrane conductance regulator; CT, carboxyl-terminus; ER, endoplasmic reticulum; GST, glutathione-S-transferase; HA, hemagglutinin; His, hexahistidinetagged; mGluR, metabotropic glutamate receptor; NHERF-2, Na + /H + exchanger regulatory factor-2; PDZ, PSD95/Discslarge/ZO1homology; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; siRNA, small interfering RNA. AbstractIn this study, we investigated the association of metabotropic glutamate receptor subtype-5a (mGluR5a) with cystic fibrosis transmembrane conductance regulator-associated ligand (CAL). Using glutathione-S-transferase pull-down techniques, we found that mGluR5a directly interacted with CAL, with the C-terminus of the receptor binding to the PSD95/Discslarge/ ZO-1 homology domain of CAL. The last four amino acids (S-S-S-L) of the C-terminus of the receptor were essential determinants for the interaction. Co-immunoprecipitation experiments and immunofluorescence assays revealed that full-length mGluR5a also associated with intact CAL in vivo, an observation consistent with the results from studies on fragment interactions in vitro. Functionally, upon co-expression with mGluR5a, CAL profoundly inhibited the ubiquitination of mGluR5a and enhanced receptor expression at the protein level but not at the mRNA level. These findings reveal that mGluR5a protein expression is physiologically regulated via its interaction with CAL. These results also suggest a molecular mechanism by which mGluR5a protein expression may be regulated at the post-translational level by the CAL protein, possibly by blocking ubiquitination-dependent receptor degradation.

The Golgi-associated PDZ Domain Protein PIST/GOPC Stabilizes the β1-Adrenergic Receptor in Intracellular Compartments after Internalization

“…Previous studies with the CFTR have shown that PIST may target an associated membrane protein for lysosomal degradation (14,15). However, PIST does not appear to induce degradation of its associated G-protein-coupled receptors (6,9). In fact we report here that PIST stabilizes the ␤1 receptor and prevents postendocytic lysosomal degradation.…”

“…Whereas several receptors are sorted into a lysosomal, degradative pathway, others may be subject to recycling to the plasma membrane to allow for a new round of receptor activation and signaling. C-terminal PSD-95/discs large/ZO-1 (PDZ) ligand motifs, which enable selected receptors to bind to PDZ domain-containing proteins, have been established as important determinants for recycling (4) or degradation (6). Whereas some PDZ domain proteins serve as scaffolds for GPCR-associated signaling complexes at the plasma membrane (7,8), it is now becoming clear that receptors are handed over to other PDZ domain proteins during their passage through intracellular compartments (9).…”

“…The relevance of PIST during postendocytic sorting of receptors is so far unclear. PIST binds to GPCRs of different classes, including metabotropic glutamate receptors (6,19), the somatostatin receptor subtype 5 (9,18), and the ␤1-adrenergic receptor. As PIST does not bind to the closely related ␤2 receptor (17), this difference between two otherwise closely related receptors enabled us to assess the specific role of PIST by comparing differences between the two receptors in their biosynthetic pathway and postendocytic sorting mechanisms.…”

A Novel NHERF1 Mutation in Human Breast Cancer and Effects on Malignant Progression

“…GST pull-down assays and western blotting were performed as described previously (28). Antibody to NHERF1 was purchased from Becton Dickinson Company (Franklin Lake, NJ, USA), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control (Santa Cruz Biotechnology, Santa Cruz, CA, USA).…”

PDZ Protein Regulation of G Protein–Coupled Receptor Trafficking and Signaling Pathways

“…Additionally, CAL colocalizes with mGluR1a following agonist activation, and its overexpression decreases mGluR1a-stimulated ERK signaling (Zhang et al, 2008b). CAL is suggested to regulate mGluR5a function by increasing the expression of the receptor by a mechanism that involves the inhibition of mGluR5a ubiquitination (Cheng et al, 2010). Taken together, it appears CAL could have a regulatory role over the subcellular localization of a subset of GPCRs, perhaps by contributing to the post-translational modification of nascent and mature proteins that ultimately influence the sorting and trafficking fate.…”