We are currently investigating the biochemical properties of the ECD portions of the hCaR and two sweet taste receptors, T1R2 and T1R3. We have constructed both baculovirus and mammalian expression vectors for these gene products and have examined the cellular processing of these secreted ECD structures in mammalian cells. Our findings indicate that ER export of both the full-length and the hCaR ECD structure is modulated by two ras-homologous GTP-binding proteins, Sar-1 (a critical component of COPII-vesicles) and Rab-1 for ER to Golgi transport in various mammalian cell-lines. The data also suggest that modifications of the hCaR ECD structure with disulfide-linked dimerization and N-linked glycosylation are important quality control mechanisms that are necessary for the secretion of the hCaR ECD structure. While a correctly folded ECD of the hCaR appears to play a dominant role in ER-Golgi trafficking and secretion of the ECD structure, surprisingly, we have not found ER export signal sequences within the carboxyl-tail of the receptor known to play a role in COPII-dependent export of many transmembrane proteins. We intend to investigate whether hCaR utilizes a unique sorting pathway with an as yet unidentified export mechanism utilizing the ECD structural determinants as a cargo component. Because our findings suggest that in a family-3 GPCR the ECD may be the primary structural determinant for transport and signaling functions, we tested the secretion properties of the two sweet taste receptor ECDs of T1R2 and T1R3 in HEK293 cells. Also, as an important tool, we produced monoclonal antibodies against the human T1R2 and T1R3 ECD structures to probe the biochemical/biophysical properties of the T1R2 and T1R3 receptors. While the T1R2/T1R3 is known to function as a heterodimer in sweet taste signaling, no detailed biochemical studies are available to determine structural determinants of the receptors contributing to dimer formation or in the specificity of sweet taste signaling for a multitude of sweet substances. Our data now provide the first preliminary evidence that suggest that properly folded T1R2 ECD can be processed and secreted into the culture medium of HEK293 cells in the absence of T1R3 gene expression. We also find that N-glycosylation and probably dimerization may provide quality-control checkpoints in the ER for the ECD domain to be transported to the cell surface in mammalian cells. In the coming years, we hope to delineate the functional significance of sweet taste receptors dimerization, determine the structural determinants contributing to the specificity, and define the link between homo- and hetero-dimerization of sweet taste receptors in cell surface trafficking.
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Zhuang, Xiaolei; Chowdhury, Shoaib; Northup, John K et al. (2010) Sar1-dependent trafficking of the human calcium receptor to the cell surface. Biochem Biophys Res Commun 396:874-80 |