Best Practice & Research Clinical Endocrinology & Metabolism
2The calcium sensing receptor life cycle: Trafficking, cell surface expression, and degradation
Introduction
G protein-coupled receptors (GPCRs) are integral membrane proteins, requiring synthesis at the endoplasmic reticulum (ER), rapidly followed by interaction with quality control complexes which target improperly folded receptors to the proteasome for degradation.1, 2, 3 Successful navigation of the quality control gauntlet is followed by chaperone- and small GTP-binding protein-assisted trafficking to their site(s) of function, generally at the plasma membrane and/or endocytic compartments, where they may signal through arrestin-scaffolded complexes (reviewed in Refs. 4, 5). Exocytic trafficking of GPCRs to the cellular compartments where they function is generally rapid and complete, with only minor intracellular levels of nascent receptors at steady state (reviewed in Refs. 6, 7). Motif-based exit from the ER is required for some GPCRs, and simple, linear motifs have been identified in diverse domains including the N-terminus, which is exposed to the ER lumen, and at intracellular loops and/or the C-terminus, which are exposed to the cytoplasm.8, 9, 10, 11 Small GTP-binding proteins of the Rab family regulate trafficking of GPCRs at multiple steps in the secretory and endocytic pathways (reviewed in Refs. 12, 13). Other classes of small GTP-binding proteins contribute to anterograde trafficking of GPCRs, including the ADP-ribosylation factor family members Sar1, which is critical for the initial incorporation into coatomer protein II (COPII) vesicles at the ER,14 and ARF6, which contributes to both pre- and post-plasma membrane vesicular trafficking, actin remodeling and cell motility.15, 16, 17, 18 Activated plasma membrane-resident GPCRs undergo covalent modifications, including phosphorylation and ubiquitinylation.19, 20 Such covalent modifications of GPCRs alter protein interactions with scaffolds, including arrestins, which often reduces plasma membrane signaling but can initiate alternate signaling pathways that continue after endocytosis.4, 5 For some GPCRs, signaling specificity and subdomain targeting at the plasma membrane is promoted by assembly of signalplexes in the ER which co-traffic to their site(s) of function.21, 22 GPCRs have been shown to signal directly to the nucleus when targeted (by as yet unknown mechanisms) to microdomains of the plasma membrane which invaginate to juxtanuclear regions.23 Some GPCRs have recently been shown to target directly to the nuclear membrane, leading to signaling unique from that initiated at the plasma membrane.24, 25, 26 Finally, targeting of GPCRs, including the cannabinoid 1 receptor, to the mitochondrial membrane by specific mitochondrial targeting sequences has recently been shown to alter neuronal mitochondrial metabolism.27 Overall, understanding of anterograde trafficking of GPCRs from the ER to their site(s) of action has lagged behind characterization of the endocytic branch of GPCR life cycles. The focus of this chapter is the CaSR, a Class C GPCR activated by extracellular Ca2+ and polycations. A detailed understanding of the compartment-specific protein chaperones and regulatory mechanism(s) that contribute to CaSR trafficking through the cell is still evolving. Fig. 1 illustrates the life cycle of CaSR, from synthesis to degradation, which will form the focus of this article. Illustrated are the interactions which are currently known to contribute to CaSR trafficking, desensitization and/or degradation, and in subsequent sections I will highlight what is known and, more importantly, outline the remaining challenges.
Section snippets
Physiological properties of the CaSR hinting at unique aspects of regulation
CaSR is among a small group of GPCRs that must function in the chronic presence of agonist. The extracellular domain (ECD), which binds Ca2+ at multiple sites,28, 29 is exposed to organellar Ca2+ during transit through the secretory pathway, and to extracellular Ca2+ (Ca2+o) at the plasma membrane. Despite the constant presence of agonist, CaSR undergoes weak functional desensitization in most studies, and continues to elicit Ca2+i responses as long as elevated Ca2+o and/or positive allosteric
Agonist-driven insertional signaling (ADIS)
CaSR signaling is dynamically regulated by agonist-evoked trafficking of nascent CaSR through the secretory pathway.30, 37, 38 Continuous elevation of Ca2+o or addition of allosteric activators induces a net increase in the steady state level of plasma membrane-localized CaSR. The net increase in plasma membrane CaSR results predominantly from an increase in anterograde trafficking through the secretory pathway (rates k1 and k2 of Fig. 1), at a constant rate of endocytosis (rate k3 of Fig. 1).30
Proteins facilitating CaSR transitions between organellar compartments
CaSR undergoes initial ER quality control (ERQC) during the cotranslational and immediate post-translational period (reviewed in Ref. 40). Exit of the CaSR from the ER is orchestrated by interactions with proteins at ER exit sites, including p24A (transmembrane emp24 domain trafficking protein 2 (TMED2)),41 a member of the family of ≈24 kDa type 1 transmembrane proteins that function as cargo receptors in the secretory pathway (reviewed in Ref. 42). p24A is predominantly localized in the early
Maintenance of an intracellular reservoir of CaSR
Immunostaining and immunohistochemistry of endogenous CaSRs in many cell types indicate that the dominant location of cellular CaSR is in pre-plasma membrane compartments (e.g., Refs. 51, 52, 53, 54, 55). Similar results are observed when CaSR is heterologously expressed in a range of cell types. The predominantly pre-plasma membrane localization of CaSR has also been confirmed using [35S]-cysteine pulse-chase methods. [35S]CaSR exhibits slow remodeling of glycosylation, i.e., less than 50% of [
Localization of CaSRs to plasma membrane micro-domains
Polarized cells express CaSRs in specific domains, and the mechanism(s) and/or protein interactions contributing to such subcellular targeting are largely unknown. Of particular functional interest is its differential targeting in distinct nephron segments, to localize apically, basolaterally, or with similar levels of expression in both membranes (reviewed in Refs. 64, 65). The CaSR is also localized apically in ductal cells of salivary glands66 and the exocrine pancreas,67 supporting a role
Mechanisms regulating endocytosis of CaSR
Endocytosis of GPCRs is a multi-step process, generally beginning with phosphorylation by G protein-coupled receptor kinases (GRKs) or other signaling-activated protein kinases, leading to incorporation of the phosphorylated GPCR into endocytic vesicles. These vesicles are either recycled to the plasma membrane after resensitization of the GPCR, or directed to lysosomes for degradation via the multivesicular body (MVB).20, 84 CaSR endocytosis is facilitated by GRKs and protein kinase C, and
Lysosomal targeting and degradation
CaSR proteins are degraded by proteasomes or lysosomes, and the mechanism(s) that target receptors to these two degradative pathways are emerging. Ubiquitination targets membrane proteins to the proteasome and requires the successive actions of three enzymes. The E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzymes are common to many targets. Specificity is conferred primarily by members of the E3 ligase family, which catalyze the final step of the pathway.89 The CaSR is
Alterations of CaSR trafficking caused by mutations
Several disorders of calcium metabolism result from CaSR mutations (reviewed in Ref. 96), with various loss-of-function mutations causing familial hypocalciuric hypercalcemia (FHH) or, when present in both alleles, neonatal severe hyperparathyroidism (NSHPT). Loss-of-function mutations can alter trafficking and/or plasma membrane targeting, causing retention in intracellular, pre-plasma membrane compartment(s).40, 56, 97, 98, 99 Pharmacochaperones including the clinical agent cinacalcet can
Summary
The life cycle of the CaSR is complex, and contributes directly to a unique feature of CaSR signaling, i.e., minimal functional desensitization despite the chronic presence of Ca2+o. There is strong evidence for a significant pool of intracellular CaSR that can be mobilized to the plasma membrane upon initiation of signaling. Anterograde trafficking of CaSR utilizes both common (Sar1, Rab and ARF GTP-binding proteins) and specific (p24A, RAMPs, 14-3-3, calmodulin) partners, and various
Acknowledgments
I thank the many lab members who have contributed to our evolving understanding of CaSR trafficking, and acknowledge Geisinger Clinic for support.
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