The extracellular calcium-sensing receptor belonging to the same family3 as the sweet and amino acid sensing T1R receptors and it has recently been found to be involved in kokumi taste enhancing perception. A metabotropic glutamate receptor belonging in this class also participates in umami taste. We have initiated studies to understand cellular processing of the family 3 GPCRs. Heterologous cell expression of family 3 GPCRs has been one of the major bottlenecks in developing functional screening strategies for these receptors. Using human calcium-sensing receptor (hCaR) as a model, we tested the sequence elements in the hCaR required for cell surface expression. Our previous studies had focused on the cell-sorting pathway(s) involved in transporting the newly synthesized hCaR from the ER to the cell surface, and we had identified a novel Rab1/Sar1 dependent mechanism recognizing the N-terminal extraceullar ligand binding domain for transport. In the present studies we examined sequence in the C-terminal domain know to modulate cell-surface expression of the hCaR. A large naturally-occurring carboxyl terminus deletion between residues S895 to V1075 of the hCaR causing autosomal dominant hypocalcemia (ADH) showed gain-of-function and increased cell surface expression in HEK-293 cells. To identify the underlying mechanism(s) for this increase, we investigated carboxyl-tail truncation and deletion hCaR mutants using a combination of biochemical and cell imaging approaches to define essential motifs regulating surface expression. The data indicate the existence of rapid constitutive receptor internalization of hCaR, accumulation in early (Rab7 positive) and late endosomal (LAMP-1 positive) sorting compartments, before lysosomal degradation. Recycling of receptors back to the cell surface was also evident. Deletion analysis has defined a 53 residue sequence required for targeting to lysosomes for degradation but not for internalization or recycling of the receptor. No single sequence motif was identified in our studies. This interval includes a high proportion of acidic and hydroxylated amino acid residues in this interval suggesting a similarity to a PEST-like degradation motif. The results define a novel large PEST-like sequence that participates in the sorting of internalized hCaR routed to the lysosomal degradation pathway that regulates cell surface abundance of the receptor (manuscript in preparation)
| Zhuang, Xiaolei; Northup, John K; Ray, Kausik (2012) Large putative PEST-like sequence motif at the carboxyl tail of human calcium receptor directs lysosomal degradation and regulates cell surface receptor level. J Biol Chem 287:4165-76 |
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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 |
