The goal of this proposal is to understand the molecular mechanism for GPCR localization in polarized target cells. Using MDCK cells as a model system, the PI has shown that the alpha 2A and 2C adrenergic receptors (ARs) are directly delivered to and retained on the basolateral surface while A1-ARs are predominantly localized (65-85%) on the apical surface and the alpha 2B-AR achieves basolateral localization by random delivery and preferential retention on that surface. The PI has also been able to eliminate several possible motifs such as the TM7 NPVIYTIF sequence, glycosylation, the i3 loop (delta 241-360) and fatty acid acylation as well as the requirement for functional coupling to G proteins as potential localization signals for the alpha 2A-AR, although the deletion of most of the i3 loop resulted in increased rate of loss of the alpha 2A-AR from the basolateral surface. There are three specific aims. The first is to use chimeric alpha 2A-AR/A1-AdR or alpha 2B-ARs to determine subdomains that are critical for direct basolateral, as opposed to apical, localization.
The second aim utilizes several approaches including the yeast two-hybrid system, lamba-zap expression libraries, gel overlays, co-immuno-isolation and/or reversible cross-linking, and co-purification with loop i3-GST fusion proteins to identify proteins that interact with cytosolic domains of the alpha 2A, 2B and 2C ARs and may play a role in their localization and/or signaling.
The final aim i s to use permeabilized MDCK cells expressing epitope-tagged alpha 2A or A1-AdR, targeting-competent cell domains or targeting-defective chimeras, to identify interacting proteins in the trans golgi network (TGN) or its budded-off vesicles by cross-linking and immunoprecipitation that play a role in selective targetting. An alternative approach is to first isolate the TGN of MDCK cells and use epitope-tagged alpha 2-AR subtype and hexa-his tagged A1-AdR to co-purify associated proteins or associated proteins trapped with reversible cross-linkers. Taken together, these results should yield information that increases our knowledge of the components and mechanisms involved in GPCR localization.
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