The present studies propose to examine what structural domain(s) within the alpha2-adrenergic receptor (alpha2AR) are responsible for targeting and/or retention of this receptor on the basolateral membrane of renal epithelial cells. The Madin-Darby canine kidney (MDCKII) cell line will be the primary model system for these studies. Site and deletion mutagenesis strategies will be employed to explore whether N-terminal glycosylation, secondary structure within the large third cytoplasmic loop, endocytosis signals located at the base of predicted transmembrane helix 7, endofacial aromatic residues and/or acylation of the alpha2AR play a critical role in alpha2AR polarization in MDCKII cells. Permanent transformants of MDCKII cells expressing genes coding for wild-type and mutant alpha2AR will be cloned and characterized for alpha2AR expression. Polarization of the MDCKII cells will be achieved by growth on permeable supports (Transwell culture wells) and alpha2AR distribution monitored using three independent strategies, including 1) biotinylation/extraction/streptavidin fractionation, 2) morphological localization and 3) cell surface ELISA techniques. The studies proposed will provide novel insights into the molecular basis for targeting of alpha2AR in renal epithelia that likely will reflect structural features exploited by all GTP-binding protein-coupled receptors for localization to specialized cellular domains. We will extend our findings in renal epithelial cells by examining whether or not similar structural domains target the alpha2AR to the basolateral domain of intestinal epithelial polarized in culture. Finally, future studies informed by the proposed experiments hopefully will reveal whether or not basolateral targeting/retention signals within varying alpha2AR subtypes determine receptor delivery to discrete regions within neurons, e.g., the somatodendritic versus synaptic terminal membranes.
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