Recombinant Human Gastrin/cholecystokinin type B receptor (CCKBR)

What is the genomic organization of the human CCKBR gene?

The human gastrin/CCKB receptor gene contains a 1356-bp open reading frame consisting of five exons interrupted by four introns and is assigned to human chromosome 11p15.4. A region of exon 4, which encodes a portion of the putative third intracellular loop, appears to be alternatively spliced to yield two different mRNAs. The long isoform (452 amino acids) contains the pentapeptide sequence Gly-Gly-Ala-Gly-Pro, while the short isoform (447 amino acids) lacks this sequence. These receptor isoforms may contribute to functional differences in gastrin- and CCK-mediated signal transduction .

How does CCKBR distribution differ between tissues?

CCKBR is a type B gastrin receptor with high affinity for both sulfated and nonsulfated CCK analogs. It is found principally in the central nervous system and the gastrointestinal tract. In the stomach, CCKBR expression has been identified in parietal cells and chromogranin A+ (ChgA+) ECL cells in the corpus, but not in gastric intrinsic factor+ (GIF+) chief cells. In the gastric antrum, CCKBR+ cells are frequently detected as single cells positioned just above the Lgr5+ cells at the base of the gland .

What methodologies are recommended for measuring CCKBR ligands in biological samples?

For accurate measurement of gastrin and CCK, specific radioimmunoassays are recommended to avoid cross-reactivity issues. For gastrin measurement, an antiserum (no. 2604) directed against the C-terminus of gastrin-17 can be used, which binds all C-terminally amidated gastrins (-14, -17, -34, -52 and -71) with equimolar potency but does not cross-react with CCK. For CCK measurement, an antiserum (no. 92128) that binds bioactive forms of CCK (-8, -22, -33 and -58) with equal potency without cross-reactivity with gastrin is recommended. Quality control is essential; in reference studies, the coefficient of variation for these assays was 32% for gastrin and 23% for CCK .

How does CCKBR contribute to gastric epithelial stem cell identity?

CCKBR defines antral stem cells at position +4, which overlaps with an Lgr5neg or low cell population but is distinct from typical antral Lgr5high stem cells. Using CCK2R-CreERT mice crossed with reporter mice for lineage tracing experiments, researchers have demonstrated that CCK2R+ recombined cells expand rapidly to trace whole antral glands within 10 days after induction, with traced glands persisting beyond 12 months. This indicates that CCK2R labels antral stem cells with long-term self-renewal capacity .

What techniques can be used to isolate and culture CCKBR+ gastric stem cells?

CCKBR+ gastric stem cells can be isolated using fluorescence-activated cell sorting (FACS) from CCK2R-CreERT reporter mice. These cells can then be cultured in a three-dimensional in vitro system that supports the growth and differentiation of gastric organoids. This system typically includes growth factors such as EGF, Noggin, and R-spondin. For experimental manipulation of CCK2R signaling, researchers can utilize selective CCK2 agonists (e.g., Gastrin I human, Caerulein) or antagonists (e.g., LY 288513, L-365,260, YM 022, CI 988) .

How do progastrin and CCK2R interact to regulate gastric stem cell dynamics?

Progastrin, an incompletely cleaved precursor of gastrin secreted by G-cells in the gastric antrum, can interconvert Lgr5neg or low CCK2R+ cells into Lgr5high cells. This conversion has been demonstrated in Lgr5-GFP;hGAS (human progastrin-overexpressing) double transgenic mice, where the expression of CCK2R in the Lgr5-GFP+ population is markedly increased. This suggests that progastrin can activate and expand CCK2R+ stem cells, potentially contributing to gastric gland homeostasis and carcinogenesis. Importantly, this interconversion is specific to undifferentiated CCK2R+ stem cells, as ChgA+ endocrine cells do not become Lgr5-positive even with progastrin overexpression .

What are the implications of CCKBR alternative splicing in gastric cancer research?

Alternative splicing of CCKBR, particularly in exon 4 encoding the third intracellular loop, results in two receptor isoforms that may have different signaling properties. A misspliced transcript variant, including an intron, has been observed specifically in colorectal and pancreatic tumor cells. Research should consider which isoform is being studied, as these structural differences may contribute to functional variations in gastrin- and CCK-mediated signal transduction. When designing experiments to target CCKBR in cancer cells, researchers should account for potential isoform-specific responses to agonists and antagonists .

How can CCKBR lineage tracing be optimized for gastric stem cell research?

For optimal lineage tracing of CCKBR+ cells, researchers should consider the following methodological approach:

• Generate CCK2R-CreERT mice using BAC transgenesis to ensure faithful recombination that reflects the endogenous expression pattern

• Cross with appropriate reporter mouse lines (e.g., Rosa26-LSL-tdTomato)

• Optimize tamoxifen dosing (1-6 mg has been successful, with lower doses reducing potential toxicity)

• Analyze traced cells at multiple time points (24 hours to 12+ months)

• Perform co-immunostaining with markers for differentiated cell types to assess the multilineage differentiation potential

This approach has successfully demonstrated that CCK2R+ cells can trace whole antral glands that persist long-term, confirming their stem cell identity .

What is the epidemiological evidence linking CCKBR signaling to gastric cancer risk?

Studies have shown that elevated serum levels of gastrin, a CCKBR ligand, are associated with increased risk of gastric non-cardia adenocarcinomas (GNCA). In a case-control study nested within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study, individuals with high gastrin levels (Q4 vs Q1) had a significantly increased risk of GNCA (fully adjusted OR: 1.92; 95% CI: 1.21, 3.05). This association became stronger after additional adjustment for CCK (Q4 vs Q1: OR: 2.56; 95% CI: 1.52, 4.30). Furthermore, serum gastrin was significantly associated with risk of gastric carcinoids (age-adjusted continuous model OR: 4.67; 95% CI: 2.67, 8.15), though the number of carcinoid cases was small. The role of CCK in cancer risk appears less clear, with inconsistent associations observed .

How does progastrin-CCKBR signaling contribute to gastric carcinogenesis?

Progastrin appears to promote gastric carcinogenesis through several CCKBR-dependent mechanisms:

• Interconversion of Lgr5neg or low CCK2R+ cells into Lgr5high cells

• Expansion of CCK2R+ cell numbers

• Promotion of gland fission

• Enhancement of carcinogenesis in response to chemical carcinogens (e.g., MNU)

These effects can be attenuated by pharmacological inhibition or genetic ablation of CCKBR, suggesting that progastrin-CCKBR signaling represents a potential therapeutic target for gastric cancer prevention and treatment. Researchers should consider these mechanisms when designing studies to investigate gastric carcinogenesis and when developing targeted therapies .

What experimental models are recommended for studying CCKBR-mediated gastric carcinogenesis?

The following experimental models have proven valuable for investigating CCKBR in gastric cancer:

Combined approaches using these models provide complementary insights into CCKBR function in normal and malignant gastric tissue .

How can researchers distinguish between CCKBR and CCKAR in experimental settings?

To distinguish between CCKBR (CCK2R) and CCKAR (CCK1R) in experimental settings, researchers should consider:

• Using selective agonists:

  • Gastrin I (human) as a selective CCK2 agonist

  • Caerulein as a CCK agonist that acts on both receptors

• Using selective antagonists:

  • LY 288513, L-365,260, YM 022, and CI 988 as selective CCK2 antagonists

  • Specific CCK1R antagonists for comparison studies

• Employing molecular techniques:

  • Receptor-specific antibodies for immunohistochemistry and Western blotting

  • Specific qPCR primers for distinguishing receptor subtypes

  • CRISPR-Cas9 targeting specific receptor genes for knockout studies

These approaches help ensure that observed effects are attributed to the correct receptor subtype .

What considerations should be made when designing radioimmunoassays for CCKBR ligands?

When designing radioimmunoassays for CCKBR ligands, researchers should consider:

• Cross-reactivity: Since gastrin and CCK comprise a family of gastrointestinal hormones that share an identical carboxyl-terminal pentapeptide sequence, it has been historically difficult to measure CCK and avoid cross-reactivity with gastrin. Use highly specific antisera: antiserum no. 2604 for gastrin and antiserum no. 92128 for CCK.

• Quality control: Include blinded quality control samples (10% of total samples) from a single serum pool within each batch to calculate coefficients of variation.

• Sample volume considerations: Prioritize measurements based on sample volume availability.

• Detection of multiple forms: Ensure the assay detects all bioactive forms of the hormone (for gastrin: -14, -17, -34, -52 and -71; for CCK: -8, -22, -33 and -58).

• Statistical analysis: Account for batch effects and interassay variability when analyzing results .

What are the key considerations when interpreting CCKBR expression data across different tissues?

When interpreting CCKBR expression data across different tissues, researchers should consider:

• Cell type heterogeneity: CCKBR is expressed in specific cell populations (e.g., parietal cells, ECL cells, +4 antral stem cells) that may constitute a small fraction of the total tissue.

• Regional variations: Expression patterns differ between gastric corpus and antrum, and between gastrointestinal tract and central nervous system.

• Alternative splicing: The presence of alternatively spliced isoforms may affect detection depending on the primers or antibodies used.

• Pathological states: CCKBR expression may be altered in disease states, particularly in gastrointestinal cancers.

• Species differences: Expression patterns may differ between human samples and animal models.

Single-cell RNA sequencing, immunohistochemistry with cell type-specific markers, and in situ hybridization can help resolve these complexities and provide more accurate interpretation of CCKBR expression data .