Targeting of G protein-coupled receptors to the basolateral surface of polarized renal epithelial cells involves multiple, non-contiguous structural signals.

Truncations and chimeras of the alpha2A-adrenergic receptor (alpha2AAR) were evaluated to identify membrane domains responsible for its direct basolateral targeting in Madin-Darby canine kidney cells. An alpha2AAR truncation, encoding transmembrane (TM) regions 1-5, was first delivered basolaterally, but within minutes appeared apically, and at steady-state was primarily lateral in its immunocytochemical localization. A TM 1-5 truncation with the third intracellular loop revealed more intense lateral localization than for the TM 1-5 structure, consistent with the role of the third intracellular loop in alpha2AAR stabilization. Addition of TM 6-7 of A1 adenosine receptor (A1AdoR) to alpha2AARTM1-5 creates a chimera, alpha2AARTM1-5/A1AdoRTM6-7, which was first delivered apically, resulting either from loss of alpha2AAR sorting information in TM 6-7 or acquisition of apical trafficking signals within A1AdoRTM6-7. Evidence that alpha2AARTM6-7 imparts basolateral targeting information is revealed by the significant basolateral localization of the A1AdoRTM1-5/alpha2AARTM6-7 and A1AdoRTM1-5/alpha2AARTM6-7+i3 chimeras, in contrast to the dominant apical localization of A1AdoR. These results reveal that sequences within TM 1-5 and within TM 6-7 of the alpha2AAR confer basolateral targeting, providing the first evidence that alpha2AAR basolateral localization is not conferred by a single region but by non-contiguous membrane-embedded or proximal sequences.