title: Interaction with cystic fibrosis transmembrane conductance regulator-associated ligand (CAL) inhibits beta1-adrenergic receptor surface expression.
abstract: G protein-coupled receptors such as the beta1-adrenergic receptor (beta1AR) must be trafficked to the plasma membrane in order to bind with their extracellular ligands and regulate cellular physiology. By using glutathione S-transferase pull-down techniques, we found that the beta1AR carboxyl terminus directly interacts with the cystic fibrosis transmembrane conductance regulator-associated ligand (CAL; also known as PIST, GOPC, and FIG), a protein known to be primarily localized to the Golgi apparatus. CAL contains two predicted coiled-coil domains and one PSD-95/Discs-large/ZO-1 homology (PDZ) domain. The beta1AR carboxyl terminus (CT) binds to the PDZ domain of CAL, with the last few amino acids (ESKV) of the beta1AR-CT being the key determinants for the interaction. Mutation of the terminal valine residue resulted in markedly reduced association of the beta1AR-CT with CAL. Numerous other mutations to the ESKV motif also impaired the beta1AR-CT/CAL interaction, suggesting that this motif is close to optimal for association with the CAL PDZ domain. In cells, full-length beta1AR robustly associates with CAL, and this interaction is abolished by mutation of the terminal valine to alanine of the receptor (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. Consistent with observations that CAL is a Golgi-associated protein, overexpression of CAL reduces surface expression of beta1AR. Interaction with CAL promotes retention of beta1AR within the cell, whereas PSD-95, another beta1AR-associated PDZ domain-containing protein, competitively blocks beta1AR association with CAL and promotes receptor trafficking to the cell surface. These data reveal that CAL, a novel beta1AR-binding partner, modulates beta1AR intracellular trafficking, thereby revealing a new mechanism of regulation for beta1AR anterograde trafficking through the endoplasmic reticulum-Golgi complex to the plasma membrane.
authors.affiliation: Department of Pharmacology, Rollins Research Center, Emory University, School of Medicine, Atlanta, GA 30322, USA. jqhe53@hotmail.com
authors.forename: Junqi
authors.initials: J
authors.lastname: He
authors.forename: Michele
authors.initials: M
authors.lastname: Bellini
authors.forename: Jianguo
authors.initials: J
authors.lastname: Xu
authors.forename: Amanda M
authors.initials: AM
authors.lastname: Castleberry
authors.forename: Randy A
authors.initials: RA
authors.lastname: Hall
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D000077614:Golgi Matrix Proteins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018342:Receptors, Adrenergic, beta-1 / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C505032:CFTR protein, human; D002352:Carrier Proteins; C434484:GOPC protein, human; D000077614:Golgi Matrix Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D018342:Receptors, Adrenergic, beta-1; D019005:Cystic Fibrosis Transmembrane Conductance Regulator