Jeffrey R. Keefer

Receptors Coupled to Pertussis Toxin-sensitive G-proteins Traffic to Opposite Surfaces in Madin-Darby Canine Kidney Cells A1 ADENOSINE RECEPTORS ACHIEVE APICAL AND α2A ADRENERGIC RECEPTORS ACHIEVE BASOLATERAL LOCALIZATION

The α2A adrenergic receptor (α2AAR) previously was shown to be directly delivered to and retained on the lateral subdomain of renal epithelial cells. The present studies demonstrate that, in contrast, wild-type and epitope-tagged canine A1 adenosine receptors (A1AdoR) are apically enriched (65-83%) in Madin-Darby canine kidney (MDCKII) and porcine renal epithelial (LLC-PKI) cells, based on surface biotinylation strategies detecting photoaffinity-labeled A1AdoR. Confocal microscopy corroborated the apical enrichment of the epitope-tagged A1AdoR. Metabolic labeling studies revealed that this steady-state polarization is achieved by direct delivery to both the apical (60-75%) and basolateral surface. Growth of A1AdoR-expressing cells as monolayers was achieved only following Transwell culture in the presence of A1AdoR antagonists, which decreased cell growth, suggesting that A1AdoR elicit MDCKII cell proliferation. The preferential apical but detectable basolateral localization of A1AdoR provides a molecular understanding of published reports that functional responses can be elicited following apical as well as basolateral delivery of adenosine agonists in varying renal preparations. These findings also suggest that receptor chimeras derived from the Gi/Go-protein-coupled α2AAR and A1AdoR will be informative in revealing structural features critical for basolateral versus apical targeting.

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 α2A-adrenergic receptor (α2AAR) were evaluated to identify membrane domains responsible for its direct basolateral targeting in Madin-Darby canine kidney cells. An α2AAR 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 α2AAR stabilization. Addition of TM 6–7 of A1 adenosine receptor (A1AdoR) to α2AARTM1–5 creates a chimera, α2AARTM1–5/A1AdoRTM6–7, which was first delivered apically, resulting either from loss of α2AAR sorting information in TM 6–7 or acquisition of apical trafficking signals within A1AdoRTM6–7. Evidence that α2AARTM6–7 imparts basolateral targeting information is revealed by the significant basolateral localization of the A1AdoRTM1–5/α2AARTM6–7 and A1AdoRTM1–5/α2AARTM6–7+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 α2AAR confer basolateral targeting, providing the first evidence that α2AAR basolateral localization is not conferred by a single region but by non-contiguous membrane-embedded or proximal sequences.

Differential targeting and retention of G protein-coupled receptors in polarized epithelial cells.

Localization of receptors in discrete cellular microdomains undoubtedly contributes to their interaction with particular effectors and receptor targets. For G protein-coupled receptors, virtually nothing is known about the mechanisms and structural features responsible for their targeting to and retention in varying surface domains. We have shown that the Gi/ Go-coupled alpha 2A-adrenergic receptor (alpha 2AAR) is directly targeted to the lateral subdomain of MDCK II cells. Mutational analysis has revealed that regions in or near the bilayer are likely critical for alpha 2AAR targeting, whereas endofacial domains contribute to alpha 2AAR retention on the lateral surface. Although the alpha 2BAR also is enriched on the lateral subdomain at steady-state, its polarization occurs after initial random delivery to both apical and basolateral surfaces followed by a selective accumulation on the lateral subdomain. The alpha 2CAR also is expressed on the lateral subdomain and achieves its localization via direct delivery to the basolateral surface; however, the alpha 2CAR also exists in an as yet not fully characterized intracellular compartment. Interestingly, another Gi/Go-coupled receptor, the A1 adenosine receptor, is enriched on the apical surface of MDCK II calls and achieves this localization by direct apical delivery. These findings indicate that receptor delivery to polarized surfaces is not determined by receptor coupling to a specific subpopulation of G proteins.

Morphological and biochemical strategies for monitoring trafficking of epitope-tagged G protein-coupled receptors in agonist-naive and agonist-occupied states

Epitope tagged alpha 2-AR subtypes have been used to address a variety of cell biological questions, and the strategies used are readily applicable to all GPCR as well as other cell surface proteins. We have provided detailed protocols for successful utilization of the epitope-tagged receptor in the studies of protein localization and trafficking in epithelial cells, and the mechanisms by which this is achieved. We have also described reversible biotinytion strategies to examine agonist-dependent (and independent) receptor turnover at the cell surface.