Title

Trafficking and turnover of the calcium sensing receptor

Date of Award

12-2006

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

Gerda E. Breitwieser

Keywords

Calcium sensing, Endoplasmic reticulum, Ubiquitination

Subject Categories

Molecular Biology

Abstract

The calcium sensing receptor (CaR), a member of G protein-coupled receptor family C, regulates systemic calcium homeostasis by activating G q - and G i -linked signaling in the parathyroid, kidney and intestine.

To examine agonist-mediated cell surface trafficking of CaR, we quantified CaR by ELISA (enzyme-linked immunosorbent assay), and found that upon stimulation by extracellular Ca 2+ , CaR undergoes multiphasic internalization that is dependent on changes in intracellular Ca 2+ .

To define the importance of the intracellular C-terminus of CaR in trafficking and targeting, we characterized amino acids/regions potentially critical for receptor localization. An RKR motif (from amino acid 896 to 898) within the proximal region of the C-terminus of CaR was demonstrated to be an ER retention/retrieval signal that becomes functional when exposed. By grafting the RKR motif to ectopic sites within the C-terminus, we found that the RKR motif functions in a distance-sensitive manner, being more potent at distal locations within the C-terminus.

To further characterize the contribution of the C-terminus of CaR to trafficking and turnover, we carried out yeast two-hybrid library screening to identify potential interactors for the intracellular C-terminus of CaR. Screening both human kidney and brain cDNA libraries, we identified nineteen interacting proteins, one of which is an E3 ubiquitin ligase named dorfin ( do uble R ING- f inger prote in ). Biochemical analyses demonstrated that CaR and dorfin interact in mammalian cells; ubiquitination of CaR is observed in the presence of proteasomal inhibitor MG132; and ubiquitination and degradation of CaR are mediated by dorfin via ERAD ( e ndoplasmic r eticulum- a ssociated d egradation) pathway.

To begin to characterize the biogenesis of CaR, we examined the structural and functional checkpoints in the ER ( e ndoplasmic r eticulum) quality control mechanism for CaR. Impaired N-linked glycosylation or disulfide bond formation of CaR results in ER retention and degradation by ERAD, suggesting that glycosylation and disulfide bond formation serve as structural checkpoints in the biogenesis of CaR. Interestingly, we identified a potential conformation-dependent checkpoint in the biogenesis of the CaR, using both receptor mutants and allosteric drugs.

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