A single lys residue on the first intracellular loop modulates the endoplasmic reticulum export and cell-surface...

Export from the endoplasmic reticulum (ER) represents an initial step in intracellular trafficking of G protein-coupled receptors (GPCRs). However, the underlying molecular mechanisms remain poorly understood. We have previously demonstrated that a highly conserved Leu residue on the first intracellular loop (ICL1) is required for exit of several GPCRs from the ER. Here we found that, in addition to Leu64 residue in the ICL1, the neighboring positively charged residue Lys65also modulates the cell-surface transport of α(2A)-adrenergic receptor (α(2A)-AR). Mutation of Lys65 to Ala, Glu and Gln significantly attenuated, whereas mutation of Lys65 to Arg strongly augmented α(2A)-AR expression at the cell surface. Consistent with the effects on the cell-surface expression of α(2A)-AR, mutation of Lys65 to Ala and Arg produced opposing effects on α(2A)-AR-mediated ERK1/2 activation. Furthermore, confocal microscopy revealed that the α(2A)-AR mutant K65A displayed a strong intracellular expression pattern and was extensively co-localized with the ER marker DsRed2-ER, suggestive of ER accumulation. These data provide the first evidence indicating an important function for a single Lys residue on the ICL1 in the ER export and cell-surface expression of α(2A)-AR. These data also suggest that the ICL1 may possess multiple signals that control the cell-surface targeting of GPCRs via distinct mechanisms.