2
The calcium sensing receptor life cycle: Trafficking, cell surface expression, and degradation

https://doi.org/10.1016/j.beem.2013.03.003Get rights and content

The calcium-sensing receptor (CaSR) must function in the chronic presence of agonist, and recent studies suggest that its ability to signal under such conditions depends upon the unique mechanism(s) regulating its cellular trafficking. This chapter will highlight the evidence supporting an intracellular endoplasmic reticulum-localized pool of CaSR that can be mobilized to the plasma membrane by CaSR signaling, leading to agonist-driven insertional signaling (ADIS). I summarize evidence for the role of small GTP binding proteins (Rabs, Sar1 and ARFs), cargo receptors or chaperones (p24A, RAMPs) and interacting proteins (14-3-3 proteins, calmodulin) in anterograde trafficking of CaSR, and discuss the potential signaling specializations arising from CaSR interactions with caveolins or Filamin A/Rho. Finally, I summarize current knowledge about CaSR endocytosis and degradation by both the proteasome and lysosome, and highlight recent studies indicating that defective trafficking of CaSR or interacting protein mutants contributes to pathology in disorders of calcium homeostasis.

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.

References (100)

  • D.J. Dupre et al.

    Seven transmembrane receptor core signaling complexes are assembled prior to plasma membrane trafficking

    Journal of Biological Chemistry

    (2006)
  • V. Kumar et al.

    Activated nuclear metabotropic glutamate receptor mGlu5 couples to nuclear Gq/11 proteins to generate inositol 1,4,5-trisphosphate-mediated nuclear Ca2+ release

    Journal of Biological Chemistry

    (2008)
  • Y. Huang et al.

    Identification and dissection of Ca2+-binding sites in the extracellular domain of Ca2+-sensing receptor

    Journal of Biological Chemistry

    (2007)
  • L. Gama et al.

    A carboxyl-terminal domain controls the cooperativity for extracellular Ca2+ activation of the human calcium sensing receptor. A study with receptor-green fluorescent protein fusion

    Journal of Biological Chemistry

    (1998)
  • S. Miedlich et al.

    Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations

    Journal of Biological Chemistry

    (2002)
  • Y. Huang et al.

    Regulation of stability and trafficking of calcium-sensing receptors by pharmacologic chaperones

    Advances in Pharmacology

    (2011)
  • A. Stepanchick et al.

    The cargo receptor p24A facilitates calcium sensing receptor maturation and stabilization in the early secretory pathway

    Biochemical and Biophysical Research Communications

    (2010)
  • W. Luo et al.

    p24A, a Type I transmembrane protein, controls ARF1-dependent resensitization of protease-activated receptor-2 by influence on receptor trafficking

    Journal of Biological Chemistry

    (2007)
  • X. Zhuang et al.

    Sar1-dependent trafficking of the human calcium receptor to the cell surface

    Biochemical and Biophysical Research Communications

    (2010)
  • C.L. Tu et al.

    The role of the calcium sensing receptor in regulating intracellular calcium handling in human epidermal keratinocytes

    Journal of Investigative Dermatology

    (2007)
  • N. Chattopadhyay et al.

    Calcium-sensing receptor in rat hippocampus: a developmental study

    Developmental Brain Research

    (1997)
  • J.I. Bruce et al.

    Molecular and functional identification of a Ca2+ (polyvalent cation)-sensing receptor in rat pancreas

    Journal of Biological Chemistry

    (1999)
  • A. Cavanaugh et al.

    Calcium-sensing receptor biosynthesis includes a cotranslational conformational checkpoint and endoplasmic reticulum retention

    Journal of Biological Chemistry

    (2010)
  • Y. Huang et al.

    Calmodulin regulates Ca2+-sensing receptor-mediated Ca2+ signaling and its cell surface expression

    Journal of Biological Chemistry

    (2010)
  • M. Bai et al.

    Protein kinase C phosphorylation of threonine at position 888 in Ca2+o-sensing receptor (CaR) inhibits coupling to Ca2+ store release

    Journal of Biological Chemistry

    (1998)
  • S.H. Young et al.

    Ca2+-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor require negative feedback by protein kinase C

    Journal of Biological Chemistry

    (2002)
  • S.L. Davies et al.

    Protein kinase C-mediated phosphorylation of the calcium-sensing receptor is stimulated by receptor activation and attenuated by calyculin-sensitive phosphatase activity

    Journal of Biological Chemistry

    (2007)
  • B.C. Bandyopadhyay et al.

    Extracellular Ca2+ sensing in salivary ductal cells

    Journal of Biological Chemistry

    (2012)
  • O. Kifor et al.

    The calcium-sensing receptor is localized in caveolin-rich plasma membrane domains in bovine parathyroid cells

    Journal of Biological Chemistry

    (1998)
  • O. Kifor et al.

    m-Calpain colocalizes with the calcium-sensing receptor (CaR) in caveolae in parathyroid cells and participates in degradation of the CaR

    Journal of Biological Chemistry

    (2003)
  • C.L. Tu et al.

    The calcium-sensing receptor-dependent regulation of cell-cell adhesion and keratinocyte differentiation requires Rho and filamin A

    Journal of Investigative Dermatology

    (2011)
  • O. Rey et al.

    Amino acid-stimulated Ca2+ oscillations produced by the Ca2+-sensing receptor are mediated by a phospholipase C/inositol 1,4,5-trisphosphate-independent pathway that requires G12, Rho, filamin-A, and the actin cytoskeleton

    Journal of Biological Chemistry

    (2005)
  • S. Tharmalingam et al.

    Calcium-sensing receptor modulates cell adhesion and migration via integrins

    Journal of Biological Chemistry

    (2011)
  • R. Mamillapalli et al.

    Switching of G-protein usage by the calcium-sensing receptor reverses its effect on parathyroid hormone-related protein secretion in normal versus malignant breast cells

    Journal of Biological Chemistry

    (2008)
  • D.M. Holstein et al.

    Calcium-sensing receptor-mediated ERK1/2 activation requires Galphi2 coupling and dynamin-independent receptor internalization

    Journal of Biological Chemistry

    (2004)
  • Y. Huang et al.

    Calcium-sensing receptor ubiquitination and degradation mediated by the E3 ubiquitin ligase dorfin

    Journal of Biological Chemistry

    (2006)
  • R.J.H. Wojcikiewicz

    Regulated ubiquitination of proteins in GPCR-initiated signaling pathways

    Trends in Pharmacological Sciences

    (2004)
  • K. Ray et al.

    Evidence for distinct cation and calcimimetic compound (NPS 568) recognition domains in the transmembrane regions of the human Ca2+ receptor

    Journal of Biological Chemistry

    (2002)
  • K. Ray et al.

    Calindol, a positive allosteric modulator of the human Ca2+ receptor, activates an extracellular ligand-binding domain-deleted rhodopsin-like seven-transmembrane structure in the absence of Ca2+

    Journal of Biological Chemistry

    (2005)
  • B.K. Ward et al.

    The role of the calcium-sensing receptor in human disease

    Clinical Biochemistry

    (2012)
  • Y. Huang et al.

    Rescue of calcium-sensing receptor mutants by allosteric modulators reveals a conformational checkpoint in receptor biogenesis

    Journal of Biological Chemistry

    (2007)
  • W.R. Skatch

    Cellular mechanisms of membrane protein folding

    Nature Structural & Molecular Biology

    (2009)
  • A. Ulloa-Aguirre et al.

    Targeting of G protein-coupled receptors to the plasma membrane in health and disease

    Frontiers in Bioscience

    (2009)
  • K. Araki et al.

    Protein folding and quality control in the ER

    Cold Spring Harbor Perspectives in Biology

    (2011)
  • M.T. Duvernay et al.

    A single conserved leucine residue on the first intracellular loop regulates ER export of G protein-coupled receptors

    Traffic

    (2009)
  • M.T. Duvernay et al.

    Anterograde trafficking of G protein-coupled receptors: function of the C-terminal F(X)6LL motif in export from the endoplasmic reticulum

    Molecular Pharmacology

    (2009)
  • C. Dong et al.

    ADP-Ribosylation factors modulate the cell surface transport of G protein-coupled receptors

    Journal of Pharmacology and Experimental Therapeutics

    (2010)
  • A.K. Gillingham et al.

    The small G proteins of the Arf family and their regulators

    Annual Review of Cell and Developmental Biology

    (2007)
  • D. Cho et al.

    ARF6 and GASP-1 are post-endocytic sorting proteins selectively involved in GRK- and PKC-mediated intracellular trafficking of dopamine D2 receptor in transfected cells

    British Journal of Pharmacology

    (2013)
  • A. Marchese et al.

    Ubiquitin-dependent regulation of G protein-coupled receptor trafficking and signaling

    Cellular Signalling

    (2012)
  • Cited by (0)

    View full text