• GPR56 is a functionally diverse adherent GPCR encoded by the ADGRG1 gene. Extensive RNA splicing in GPR56 transcription results in at least five different GPR56 protein subtypes that differ in function and signal signature. GPR56 interacts with a range of endogenous protein ligands and binding partners, including collagen III, tissue transglutaminase 2 (TG2), CD9/CD81, and progastrin, which function as scaffold proteins, co-receptors, or homologous ligands. More and more studies have shown that GPR56 promotes or inhibits cancer development in different cancers in a way specific to cell type and tumorigenic stage. In 2022, GPR56 was first mentioned as a therapeutic target for colorectal cancer to develop antibody-drug couplings.
• The exons 1-14 of mouse Gpr56 gene that encode the whole molecule (ATG to STOP codon), including promoter, 5’UTR and 3’UTR were replaced by human counterparts in B-hGPR56 mice. The human GPR56 expression was driven by endogenous mouse Gpr56 promoter, while mouse Gpr56 gene transcription and translation will be disrupted.
• Human GPR56 was exclusively detectable in brain of B-hGPR56 mice but not in wild-type mice.
• Human GPR56 mRNA was exclusively detectable in homozygous B-hGPR56 mice but not in wild-type mice.

Gene targeting strategy for B-hGPR56 mice. The exons 1-14 of mouse Gpr56 gene that encode the whole molecule (ATG to STOP codon), including promoter, 5’UTR and 3’UTR were replaced by human counterparts in B-hGPR56 mice. The human GPR56 expression was driven by endogenous mouse Gpr56 promoter, while mouse Gpr56 gene transcription and translation will be disrupted.

GPR56 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hGPR56 mice by flow cytometry. Brain cells were collected from wild-type C57BL/6JNifdc mice (+/+) (male, 6-week-old, n=1) and homozygous B-hGPR56 mice (H/H) (male, 5-week-old, n=1). Protein expression was analyzed with anti-human GPR56 antibody (Biolegend, 391906) by flow cytometry. Human GPR56 was exclusively detectable in brain of B-hGPR56 mice but not in wild-type mice.

Strain specific analysis of GPR56 mRNA expression in wild-type C57BL/6JNifdc mice and B-hGPR56 mice by RT-PCR. Brain RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hGPR56 mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human GPR56 primers. Mouse Gpr56 mRNA was only detectable in wild-type mice. Human GPR56 mRNA was exclusively detectable in homozygous B-hGPR56 mice but not in wild-type mice.