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The Calcitonin Gene-Related Peptide (CGRP) family comprises CGRP itself, calcitonin (CT), adrenomedullin (AM), and islet amyloid polypeptide (AMY).

The neuropeptide CGRP is a potent vasodilator produced by central and peripheral neurons. In the human body, CGRP exists in two isoforms: α-CGRP and β-CGRP. Both α-CGRP and β-CGRP act as full agonists for their respective receptors. CGRP mediates its effects through the CGRP receptor—a heterodimeric complex consisting of a G protein-coupled receptor, CALCRL, and a single-transmembrane domain protein, RAMP1. The final component required for a functional CGRP receptor is the Receptor Component Protein (RCP). It has been demonstrated that the absence of RCP impairs CGRP receptor activation—as measured by an increase in cyclic adenosine monophosphate (cAMP)—without affecting ligand binding. The primary functional unit of the receptor, CALCRL (also known as CLR or CRLR), can associate with one of three RAMPs, each of which generates a distinct receptor type. RAMPs are single-transmembrane proteins critical to receptor function, as they facilitate the translocation of the receptor complex to the plasma membrane and further confer ligand-binding specificity. CALCRL paired with RAMP1 constitutes the CGRP receptor; paired with RAMP2, it forms the AM receptor (designated AM1); and paired with RAMP3, it forms a dual CGRP/AM receptor (designated AM2). The RAMP1 subunit is responsible for the specific binding of CGRP to the CGRP receptor.

Upon ligand binding, the receptor complex interacts with G proteins; the Gαs subunit dissociates from the complex and activates adenylyl cyclase, thereby generating cAMP. Elevated intracellular cAMP concentrations stimulate cAMP-dependent signaling pathways, ultimately leading to the binding of the transcription factor cAMP response element-binding protein (CREB) to CRE promoters, which induces gene expression.

The HEK293 Human CALCRL&RAMP1 CRE-Luc Cell Line model effectively simulates the in vivo CGRP signal transduction process, the principle is illustrated in the figure below.

Figure 1. Schematic diagram of the HEK293 Human CALCRL&RAMP1 CRE-Luc Cell Line model

Figure 2. Recombinant CALCRL&RAMP1 CRE-Luc HEK293 constitutively expressing CALCRL.

Figure 3. WB of CALCRL&RAMP1 CRE-Luc HEK293.

Figure 4. Dose Response of Agonists in CALCRL&RAMP1 CRE Luc Reporter HEK293 Cells (C1).

Figure 5. Inhibtion of αCGRP/βCGRP  induced Reporter Activity by CGRP Neutralizing Antibodies in CALCRL&RAMP1 CRE-Luc HEK293(C1).

Figure 6. HTRF cAMP Assay with CALCRL&RAMP1 CRE-Luc HEK293(C1).

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed HEK293 Human CALCRL&RAMP1 CRE-Luc Cell Line complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.