Fig. 9: Contribution of orthosteric pocket residues on GPR109A mediated signaling.

a Cartoon representation of residues interacting via H-bond with niacin (yellow), acipimox (orange red), MK6892 (cyan), GSK256073 (sandy brown), and MMF (light pink). b-c cAMP response downstream of GPR109A^WT, GPR109A^R111A, and GPR109A^S179A in response to the indicated ligands was studied by GloSensor assay (b) (mean ± SEM; n = 3 independent experiments; % normalized with the minimum concentration for each ligand as 100) and NanoBiT-based βarr1 recruitment assay (c) (mean ± SEM; n = 3 independent experiments; fold normalized with the minimum concentration for each ligand as 1). d, f, h cAMP decrease studied by GloSensor assay downstream of GPR109A^WT, and mutants in response to niacin, MK6892, and GSK256073 (mean ± SEM; n = 3–5 independent experiments; n = 3 in response to niacin and MK6892 and n = 5 in response to GSK256073; % normalized with the minimum concentration for each ligand as 100). e, g, i NanoBiT-based βarr1 recruitment downstream of GPR109A^WT, and mutants in response to niacin, MK6892, and GSK256073 (mean ± SEM; n = 3–4 independent experiments; n = 3 in response to MK6892, n = 4 in response to niacin, and GSK256073; fold normalized with the minimum concentration for each ligand as 1). Bias factor for the mutant GPR109A^S179A (shown in the inset of e) was calculated using the software https://biasedcalculator.shinyapps.io/calc/ (Detailed formula is provided in methods section). The compiled data of G-protein activation and βarr1 recruitment assays (mean ± SEM, n = 3–4 independent experiments; n = 3 for cAMP response and n = 4 for βarr1 recruitment) was used for the calculation. During bias factor calculation GPR109A^WT was considered as reference and observed G-protein bias with GPR109A^S179A upon stimulation with niacin. Source data is provided as Source Data file.