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

Area of Science:

• Structural biology
• Biochemistry
• Cell biology

Background:

• G-protein-coupled receptor class C group 6 member A (GPRC6A) is a crucial nutrient and hormone sensor involved in metabolic and endocrine regulation.
• Limited cell-surface expression of human GPRC6A (hGPRC6A) has hindered structural and functional investigations.
• Previous studies identified intracellular retention issues due to insertion/deletion variants in the Intracellular Loop 3 (ICL3) of hGPRC6A.

Purpose of the Study:

• To optimize the recombinant expression of hGPRC6A for structural characterization.
• To overcome challenges associated with hGPRC6A's limited cell-surface localization.

Main Methods:

• Engineered recombinant hGPRC6A constructs by substituting the native signal peptide and modifying the ICL3 region.
• Expressed the optimized receptor in mammalian cells.
• Purified the protein using detergent solubilization and chromatography, followed by negative-staining Electron Microscopy (EM) and 2D classification.

Main Results:

• Signal peptide substitution and ICL3 modification significantly improved hGPRC6A membrane expression.
• Negative-staining EM analysis yielded well-defined particles.
• 2D class averages revealed an architecture consistent with canonical class C G-Protein-Coupled Receptors (GPCRs).

Conclusions:

• Engineering the signal peptide and ICL3 region effectively promotes cell surface expression of GPRC6A.
• This strategy facilitates the expression and purification of challenging GPCRs, including hGPRC6A.
• The established method provides a foundation for future high-resolution structural and functional studies of hGPRC6A.