Agonist-Driven Maturation and Plasma Membrane Insertion of Calcium-Sensing Receptors Dynamically Control Signal Amplitude

Abstract

Calcium-sensing receptors (CaSRs) regulate systemic calcium homeostasis in the parathyroid gland, kidney, intestine, and bone and translate fluctuations in serum calcium into peptide hormone secretion, cell signaling, and regulation of gene expression. The CaSR is a G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptor that operates in the constant presence of agonist, sensing small changes with high cooperativity and minimal functional desensitization. Here, we used multiwavelength total internal reflection fluorescence microscopy to demonstrate that the signaling properties of the CaSR result from agonist-driven maturation and insertion of CaSRs into the plasma membrane. Plasma membrane CaSRs underwent constitutive endocytosis without substantial recycling, indicating that signaling was determined by the rate of insertion of CaSRs into the plasma membrane. Intracellular CaSRs colocalized with calnexin in the perinuclear endoplasmic reticulum and formed complexes with 14-3-3 proteins. Ongoing CaSR signaling resulted from agonist-driven trafficking of CaSR through the secretory pathway. The intracellular reservoir of CaSRs that were mobilized by agonist was depleted when glycosylation of newly synthesized receptors was blocked, suggesting that receptor biosynthesis was coupled to signaling. The continuous, signaling-dependent insertion of CaSRs into the plasma membrane ensured a rapid response to alterations in the concentrations of extracellular calcium or allosteric agonist despite ongoing desensitization and endocytosis. Regulation of CaSR plasma membrane abundance represents a previously unknown mechanism of regulation that may be relevant to other receptors that operate in the chronic presence of agonist.

The GPRC6A receptor displays constitutive internalization and sorting to the slow recycling pathway

“…Constitutive internalization of CaSR has been observed in some studies (52,53), and moreover, a distinct regulatory mechanism for CaSR has been reported in which agonist stimulation triggers further insertion of receptors in the cell membrane from intracellular stores, thereby increasing the amount of receptors available for activation. This mechanism has been named agonist-driven insertional signaling (54). Finally, the ␥-aminobutyric acid B (GABA B ) receptors do not internalize in response to agonist; however, they do exhibit constitutive internalization and subsequent recycling (55)(56)(57) in line with our current observations for the GPRC6A receptor.…”

Claudin-14 Underlies Ca++-Sensing Receptor–Mediated Ca++ Metabolism via NFAT-microRNA–Based Mechanisms

“…NPS2143 will likely have a similar pharmacokinetic profile. Resetting at the level of receptor can involve regulation of its gene expression, protein trafficking, and endocytosis 34 or interaction with adaptor proteins, such as filamin A 35 and AP2S1. 36 The renal role of CaSR in calcium metabolism has been difficult to delineate owing to its effects on PTH secretion.…”

The calcium sensing receptor: from calcium sensing to signaling

“…Several proteins interact with the CaSR when the receptor exits from the ER, including p24A (transmembrane emp24 domain trafficking protein 2(TMED2)) [101]; the small GTP-binding protein Sar1 [102]; receptor-activitymodifying proteins (RAMPs) [96]; Rab1 [103]; calmodulin (CaM) [63]; 14-3-3 [104] and Dorfin [90]. p24A binds predominantly to the immature form of the CaSR and cycles between the ER, the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi membranes [105,106].…”

“…Both of them are found to facilitate the CaSR trafficking from ER to Golgi [96,103]. The 14-3-3 protein is predicted to interact with the arginine-rich domain of the CaSR ( 890 RRxxxxRKR 898 ) and may lead to the retention of the CaSR in the ER [65,104,107]. There is also a calmodulin-binding site in the C-tail of the CaSR comprising residues 874895 [47].…”

“…This phenomenon was named Agonist-Driven Insertional Signaling (ADIS) [104]. However, a number of questions raised by the ADIS mechanism remained to be explored.…”