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

Three types of amylin receptors have been identified: AMY1, AMY2, and AMY3. Amylin receptors belong to the Class B GPCR family; they exist as heterodimeric complexes formed by the association of a calcitonin receptor (CTR) with a receptor activity-modifying protein (RAMP). The CTR component of this receptor possesses the canonical GPCR structure: seven interconnected transmembrane α-helices that transduce extracellular signals into intracellular cytoplasmic signaling cascades. There are three types of RAMPs (RAMP1, RAMP2, and RAMP3), which share 31% sequence identity and possess similar basic structures. RAMPs are critical accessory proteins responsible for transporting the CTR to the cell membrane and modulating its ligand specificity; they can alter the pharmacology, function, and cellular trafficking of specific GPCRs.

In mammalian cells, activation of amylin receptors has been shown to elevate intracellular cAMP levels and is believed to be mediated by the stimulation of Gs proteins and adenylyl cyclase. In the presence of RAMPs, the binding affinity of the receptor for amylin can be significantly modified. The heterodimeric complexes formed by CTR and RAMP1, or by CTR and RAMP3, are thought to facilitate amylin binding and mediate physiological functions within the brain. Activation of AMY3 stimulates the G protein Gαs, which in turn regulates adenylyl cyclase (AC) activity, leading to a subsequent increase in cellular cAMP levels. The binding of cAMP to the regulatory subunits (R2) of Protein Kinase A (PKA) induces the dissociation of the tetrameric PKA holoenzyme, thereby activating the catalytic subunits of PKA. cAMP-activated PKA participates in the regulation of ERK1/2 activity. Triggering of the ERK1/2 pathway may lead to an uneven distribution of intracellular Ca²⁺, precipitating endoplasmic reticulum and mitochondrial dysfunction, and ultimately resulting in cell death. Furthermore, cAMP stimulation of AKT leads to the subsequent induction of the transcription factor c-Fos.

The HEK293 Human CTR(CALCR) CRE-Luc Cell Line model effectively simulates the in vivo CTR signal transduction process, the principle is illustrated in the figure below.

Figure 1. Schematic diagram of the HEK293 Human CTR(CALCR) CRE-Luc Cell Line model

Figure 2. Dose response of  Calcitonin(salmon) in CTR(CALCR) CRE-Luc HEK293 Cell (C9).

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 CTR(CALCR) 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.