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 wished to understand cellular processing of the family 3 GPCRs because heterologous cell expression of family 3 GPCRs has been one of the major bottlenecks in developing functional screening strategies for these receptors. Using the human calcium sensing receptor, hCaR, we had previously 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. Our current work has focused on the re-cycling of the hCaR. We examined sequence in the C-terminal domain known 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. Our studies revealed a 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, which have eliminated known calmodulin-binding, protein kinase C phorphorylation and lysines targeting proteosomal degradation. 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 (Zhuang et al, manuscript in revision for J. Biol. Chem.)

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National Institute on Deafness and Other Communication Disorders
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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
Charizopoulou, Nikoletta; Lelli, Andrea; Schraders, Margit et al. (2011) Gipc3 mutations associated with audiogenic seizures and sensorineural hearing loss in mouse and human. Nat Commun 2:201
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