Role of Receptor Activity Modifying Protein 1 in Function of the Calcium Sensing Receptor in the Human TT Thyroid Carcinoma Cell Line

Abstract

The Calcium Sensing Receptor (CaSR) plays a role in calcium homeostasis by sensing minute changes in serum Ca2+ and modulating secretion of calciotropic hormones. It has been shown in transfected cells that accessory proteins known as Receptor Activity Modifying Proteins (RAMPs), specifically RAMPs 1 and 3, are required for cell-surface trafficking of the CaSR. These effects have only been demonstrated in transfected cells, so their physiological relevance is unclear. Here we explored CaSR/RAMP interactions in detail, and showed that in thyroid human carcinoma cells, RAMP1 is required for trafficking of the CaSR. Furthermore, we show that normal RAMP1 function is required for intracellular responses to ligands. Specifically, to confirm earlier studies with tagged constructs, and to provide the additional benefit of quantitative stoichiometric analysis, we used fluorescence resonance energy transfer to show equal abilities of RAMP1 and 3 to chaperone CaSR to the cell surface, though RAMP3 interacted more efficiently with the receptor. Furthermore, a higher fraction of RAMP3 than RAMP1 was observed in CaSR-complexes on the cell-surface, suggesting different ratios of RAMPs to CaSR. In order to determine relevance of these findings in an endogenous expression system we assessed the effect of RAMP1 siRNA knock-down in medullary thyroid carcinoma TT cells, (which express RAMP1, but not RAMP3 constitutively) and measured a significant 50% attenuation of signalling in response to CaSR ligands Cinacalcet and neomycin. Blockade of RAMP1 using specific antibodies induced a concentration-dependent reduction in CaSR-mediated signalling in response to Cinacalcet in TT cells, suggesting a novel functional role for RAMP1 in regulation of CaSR signalling in addition to its known role in receptor trafficking. These data provide evidence that RAMPs traffic the CaSR as higher-level oligomers and play a role in CaSR signalling even after cell surface localisation has occurred.

Receptor Activity-Modifying Proteins (RAMPs): New Insights and Roles

“…Two reports have studied the interactions between the class C GPCR, the calcium-sensing receptor (CaSR) and RAMPs (26; 27). In both cases RAMP1 and RAMP3, but not RAMP2, appeared to enable CaSR cell surface expression.…”

“…Further, Desai and colleagues used siRNA to reduce RAMP1 expression in medullary thyroid carcinoma TT cells, which naturally express mRNA for both CaSR and RAMP1. RAMP1 knockdown reduced agonist (Cinacalcet)-mediated CaSR Ca 2+ signaling, showing that RAMP1 is important for CaSR receptor function in these cells (26). It will be important to determine the importance of these interactions in vivo and their relevance to the clinical use of CaSR-targeted drugs.…”

“…Therefore, any alteration in RAMP1 expression could affect CGRP or amylin activity. As more partners for RAMP1 are identified, such as the CaSR, this becomes even more relevant (26; 27). …”

Pharmacological diversity among drugs that inhibit bone resorption

“…These receptors consist of calcitonin receptorlike receptor (CLR) linked to an essential receptor activity modifying protein (RAMP) that is necessary for its full function [9]. RAMPs are being studied as potential pharmacological targets in relation to cancer [10]. CGRP is a highly potent vasodilator and antagonists are being developed for migraine [11].…”

Thyroid C-Cell Biology and Oncogenic Transformation

“…While the precise mechanism controlling calcitonin release has not clearly been elucidated, both the calcium-sensing receptor (CASR) and cholecystokinin-B receptor (gastrin receptor) are expressed in MTC cells [61–63]. In MTC cells, the functionally of CASR has recently been demonstrated to be modulated though interactions with RAMP1, which act to regulate trafficking of the receptor to the cell surface [62]. Unfortunately, these studies are complicated by the reliance on MTC tumor cell line models rather than normal C-cells.…”