Enhanced Cell Surface Expression Enables Purification and Structural Characterization of Human GPRC6A

The G-protein-coupled receptor class C group 6 member A (GPRC6A) is a member of the class C G-Protein-Coupled Receptor (GPCR) family and functions as a nutrient and hormone sensor involved in metabolic and endocrine regulation. GPRC6A localizes to the cell membrane and forms homodimers for its physiological function. However, human GPRC6A (hGPRC6A) exhibits limited cell-surface expression, hindering its structural and functional studies. Previous studies have shown that insertion/deletion variants in the Intracellular Loop 3 (ICL3) of hGPRC6A cause intracellular retention during protein expression. This study aimed to optimize the recombinant expression of hGPRC6A to enable structural characterization.

Recombinant hGPRC6A constructs were engineered by substituting the native signal peptide and modifying the ICL3 region. The optimized receptor was expressed in mammalian cells, purified using detergent solubilization and chromatography, and analyzed by negative-staining Electron Microscopy (EM) followed by Two-Dimensional (2D) classification.

Signal peptide substitution and ICL3 modification markedly improved the membrane expression of hGPRC6A. Negative-staining EM revealed well-defined particles, and 2D class averages displayed an overall architecture characteristic of canonical class C GPCRs.

We demonstrate that engineering of the signal peptide and ICL3 region promotes proper cell surface expression of GPRC6A. This strategy provides a useful approach for the expression and purification of other GPCRs that are difficult to traffic to the plasma membrane.

We established an effective expression and purification strategy for hGPRC6A that restores membrane localization and yields well-defined particles consistent with class C GPCR architecture. These results provide a foundation for future high-resolution structural and functional studies of hGPRC6A.