Interaction with cystic fibrosis transmembrane conductance regulator-associated ligand (CAL) inhibits β1-adrenergic receptor surface expression

Abstract

G protein-coupled receptors such as the β1-adrenergic receptor (β1AR) 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 β1AR 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 β1AR carboxyl terminus (CT) binds to the PDZ domain of CAL, with the last few amino acids (ESKV) of the β1AR-CT being the key determinants for the interaction. Mutation of the terminal valine residue resulted in markedly reduced association of the β1AR-CT with CAL. Numerous other mutations to the ESKV motif also impaired the β1AR-CT/CAL interaction, suggesting that this motif is close to optimal for association with the CAL PDZ domain. In cells, full-length β1AR 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 β1AR. Interaction with CAL promotes retention of β1AR within the cell, whereas PSD-95, another β1AR-associated PDZ domain-containing protein, competitively blocks β1AR association with CAL and promotes receptor trafficking to the cell surface. These data reveal that CAL, a novel β1AR-binding partner, modulates β1AR intracellular trafficking, thereby revealing a new mechanism of regulation for β1AR anterograde trafficking through the endoplasmic reticulum-Golgi complex to the plasma membrane.