| Product Description |
Recombinant CHO-K1 cells stably overexpress human receptor activity modifying protein 1 (RAMP1) and calcitonin receptor (CTR) on the surface. Amylin receptors (AMYR) are a class of special protein heterodimers, composed of a CTR combined with one of three homologous receptor activity-modifying proteins (RAMP1, RAMP2, RAMP3), forming three subtypes (AMY1, AMY2, AMY3). This cell contain high levels of G protein Gαs to couple with the receptor in downstream signaling pathways. |
| Applications |
Calcium flux assay, IP-One assay and cAMP accumulation assay. |
| Expressed Gene |
NCBI reference sequence NM_005855.1 (for RAMP1) and NM_001742.1 (for CTR); no expressed tags |
| Target Protein |
NP_005846.1 (for RAMP1) and NP_001733.1 (for CTR) |
| Host Cell |
CHO-K1 |
| Size |
Two vials of frozen cells (>1×106 per vial in 1 mL) |
| Storage |
Store cells in liquid nitrogen immediately upon receipt. Thaw and recover cells within one year from the date received. |
| Mycoplasma Status |
Negative. The mycoplasma test was performed with MycoAlert™ PLUS Mycoplasma Detection Kit (Cat. No. LT07-318, Lonza). |
| Stability |
Stable through more than 16 passages with no significant changes in assay performance or expression profile. |
| Culture Properties |
Adherent |
| Freeze Medium |
45% Ham’s F-12K (Kaighn’s) (Cat. No. 21127, Life Technologies), 45% FBS (Cat. No. 10099-141, Life Technologies), 10% DMSO (Cat. No. D2650, Sigma) |
| Complete Growth Medium |
Ham’s F-12K (Kaighn’s), 10% FBS |
| Culture Medium |
Ham’s F-12K (Kaighn’s), 10% FBS, 200 μg/ml Zeocin (Cat. No. R250-01, Life Technologies), 100 μg/ml Hygromycin B (Cat. No. 10687010, Life Technologies) |
| Gene Synonyms |
CALCR, CRT, CT-R, CTR, CTR1 (for CTR) |
| Background |
Receptor activity-modifying proteins (RAMPs) are a class of protein which interact with and modulate the activities of several Class B G Protein-Coupled Receptors including the receptors for secretin, calcitonin (CT), glucagon, and vasoactive intestinal peptide (VIP).There are three distinct types of RAMPs, designated RAMP1, RAMP2, and RAMP3, each encoded by a separate gene. Currently the function of RAMPs is divided into 2 class activities. Association of RAMPs with either the CT or CALCRL proteins forms 6 different receptors from the calcitonin receptor family. When associated with the Calcitonin receptor (CTR) or Calcitonin receptor-like (CALCRL), RAMPs can change the selectivity of the receptor for a specific hormone. In the cases of the other receptors mentioned however, there is no evidence that they can do this, but instead function to regulate trafficking of receptors from the ER / Golgi to the membrane. |
Figure 1. Calcitonin-induced concentration-dependent stimulation of intracellular calcium mobilization in CHO-K1/Gα15/AMY1 cells. The cells were loaded with Calcium-4 (Cat. No. R8142; Molecular Devices) prior to stimulation with AMY1 agonist, calcitonin. The intracellular calcium change was measured by FLIPRTETRA. The relative fluorescent units (RFU) were recorded and normalized to plot against the log of the cumulative doses of calcitonin (mean ± SEM, n = 3). The EC50 of calcitonin on CHO-K1/Gα15/AMY1 cells was 0.11 μM.
Notes:
EC50 value is calculated with four parameter logistic equation:
Y=Bottom + (Top-Bottom) / (1+10^ ((LogEC50-X)*Hill Slope))
X is the logarithm of concentration. Y is the response.
Y is RFU and starts at Bottom and goes to Top along a sigmoid curve.
Figure 2. Dose dependent stimulation of intracellular cAMP accumulation upon treatment with amylin in CHO-K1/Gα15/AMY1 cells. d2 acceptor fluorophore-labeled cAMP (Cat. No. 62AM4PEC; Revvity) and intracellular cAMP in CHO-K1/Gα15/AMY1 cells competitively bind with Europium Cryptate-labeled anti-cAMP monoclonal antibody. The FRET signal decreases as the intracellular cAMP concentration rises and was measured by plate reader (Pherastar, BMG). The EC50 of amylin on CHO-K1/Gα15/AMY1 cells was 4.94 nM.
For research use only. Not intended for human or animal clinical trials, therapeutic or diagnostic use.
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