To complement our prior examination of the signaling properties of the 7TM cores of the family 3 GPCRs (hCaR and hT1Rs), we have constructed expression vectors for the ECD portions of the hCaR, mGluR1 and hT1Rs. Attempts to express these in several E.coli strains have uniformly failed, despite co-transformation of the expression hosts with identified bacterial chaperones and thioreductase. We are now constructing baculoviral vectors for this project, and we have examined the cellular processing of family 3 GPCRs in mammalian cells. Our findings indicate that the 7TM cores and the ECD domains utilize alternate sorting pathways within HEK293 cells which can be distinguished by COPII vesicle transport from ER to Golgi using Sar1 and Rab1 GTPases. The wild-type structures for hCaR and mGluR1 appear to utilize both sorting pathways. Surprisingly, the standard ER-export signal sequence does not appear to play a role in this sorting. Further, constructs lacking the carboxyl-terminal sequences of the hCaR or ECD constructs lacking any transmembrane segments utilize a sorting pathway with an as yet unidentified cellular recognition of the extracellular sequence(s) of the proteins linking them to Sar1-Rab1 assembly (Zhuang, Northup & Ray, manuscript submitted).
| Ray, Kausik; Adipietro, Kaylin A; Chen, Claudia et al. (2007) Elucidation of the role of peptide linker in calcium-sensing receptor activation process. J Biol Chem 282:5310-7 |
