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MC2R (melanocortin 2 receptor), also known as the adrenocorticotropic hormone receptor (ACTHR), is a member of the melanocortin receptor (MCR) family. MC2R is highly specific; it is primarily expressed in the zona fasciculata and zona reticularis of the adrenal cortex, with low-level expression also found in peripheral tissues such as adipose tissue, bone, ovaries, and skin. MC2R exhibits strict ligand selectivity: unlike other family members that recognize various melanocortins (such as α-MSH), MC2R is specifically activated only by adrenocorticotropic hormone (ACTH). Clinical genetic studies have confirmed that loss-of-function mutations in the *MC2R* gene are the primary cause of familial glucocorticoid deficiency type 1 (FGD1); in these patients, adrenal cortical insensitivity to ACTH leads to severely deficient cortisol synthesis, manifesting as life-threatening adrenal insufficiency.

MC2R primarily couples with intracellular Gs proteins. Upon binding of ACTH to MC2R, the receptor undergoes a conformational change that activates the Gs protein, which in turn activates adenylyl cyclase (AC), causing a rapid increase in intracellular levels of the second messenger cyclic AMP (cAMP). cAMP subsequently activates protein kinase A (PKA); through the phosphorylation of downstream transcription factors (such as CREB) and steroidogenesis-related proteins, PKA ultimately upregulates the expression of steroidogenic acute regulatory protein (StAR) and a series of steroidogenic enzymes, thereby stimulating the efficient synthesis and secretion of cortisol by the adrenal cortical zona fasciculata.

The CHO-K1 Human MC2R CRE-Luc Cell Line model effectively simulates the in vivo MC2R signal transduction process, the principle is illustrated in the figure below.

Figure 1. Schematic diagram of the CHO-K1 Human MC2R CRE-Luc Cell Line  model

Figure 2. Dose Response of Human ACTH(1-39) in MC2R CRE-Luc CHO(C16).

Figure 3. HTRF cAMP Assay with MC2R CRE-Lue CHO(C16).

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 CHO-K1 Human MC2R 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.