Multi-predictor pathogenicity filtering and membrane-specific stability analysis prioritize and characterize candidate destabilizing missense variants of the GIP receptor (GIPR)

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is a class B G protein-coupled receptor and a validated target of incretin-based therapies for type 2 diabetes and obesity, yet the functional consequences of its missense variation remain largely uncharacterized. Because most variant-effect and stability predictors were developed for soluble, globular proteins and transfer poorly to the membrane environment, we combined multi-predictor pathogenicity filtering with membrane-specific stability analysis to prioritize the missense variants of GIPR most likely to be functionally important. Missense variants were retrieved from dbSNP, yielding 763 unique rsIDs corresponding to 893 single-nucleotide variants after multi-allelic expansion. These were filtered sequentially through PolyPhen-2, a rarity threshold based on gnomAD v4.1.1 allele frequencies, SIFT 4G, PROVEAN, AlphaMissense, MetaRNN and REVEL, and finally the membrane-specific predictor mCSM-membrane, retaining only variants classified as both pathogenic and structurally destabilizing. Fourteen rare variants met all criteria. All fourteen mapped to transmembrane helices, spanning six of the seven helices with none in TM4, and were predicted destabilizing by mCSM-membrane, with DynaMut2 concordant for ten and a second membrane-specific predictor, Missense3D-TM, calling thirteen of the fourteen structurally damaging. Structural characterization with HOPE predicted recurrent destabilizing mechanisms, including loss of core packing and a hydrogen bond for the most destabilizing variant (Y141N), introduction of a buried or lipid-exposed positive charge by five arginine substitutions, replacement of small buried glycines by valine in three variants, and disruption of the helix that presents Gln224 (Q224P), the residue that hydrogen bonds the N-terminal tyrosine of GIP in the GIP-bound receptor structure; three variants additionally contacted a bound small-molecule modulator. Together, these results provide a prioritized, mechanistically annotated set of GIPR missense variants as a foundation for future functional and pharmacogenetic studies.