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Guanylate Cyclase C (GUCY2C/GCC) belongs to the family of receptor guanylyl cyclases (GC)—enzymes capable of converting GTP into the second messenger cGMP. GUCY2C is a type I transmembrane receptor protein highly expressed on the apical membranes of intestinal epithelial cells; it serves as a receptor for the endogenous peptides guanylin and uroguanylin, as well as for the heat-stable enterotoxin produced by *E. coli*. GUCY2C is widely expressed in colorectal cancer cells and other gastrointestinal tumors. Dysregulation—specifically, either a loss or an increase in GUCY2C function—is associated with several conditions characterized by constipation or diarrhea, including Irritable Bowel Syndrome with Constipation (IBS-C), Chronic Idiopathic Constipation (CIC), and Inflammatory Bowel Disease (IBD).  

Upon activation by endogenous ligands or the exogenous bacterial toxin STa, GUCY2C catalyzes the production of the second messenger cGMP. This, in turn, regulates ion and fluid secretion in intestinal epithelial cells by activating the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), thereby maintaining fluid and electrolyte homeostasis. Simultaneously, this signaling pathway contributes to maintaining the integrity of the intestinal epithelial barrier and exerts anti-inflammatory effects, thereby protecting the gut from injury. Furthermore, GUCY2C plays a pivotal role as a tumor suppressor by inhibiting the AKT signaling pathway; it regulates the proliferation and metabolism of intestinal epithelial cells, thereby suppressing the initiation and progression of tumors such as colorectal cancer.

The HEK293 Human GUCY2C Effector Reporter Cell model accurately simulates the in vivo GUCY2C signal transduction process.

Figure 1. Schematic Diagram of the HEK293 Human GUCY2C Effector Reporter Cell Model

 
Figure 2. Recombinant GUCY2C Effector Reporter Cell stably expressing GUCY2C.


Figure 3. Dose Response of Linaclotide in GUCY2C Effector Reporter Cell (C5).

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 GUCY2C Effector Reporter Cell 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.