Biased constitutive signaling of the G protein-coupled receptor GPR35 suppresses gut barrier permeability

Agonist-independent, or constitutive, activity is an integral feature of G protein-coupled receptors, but its relevance in pathophysiological settings is generally poorly explored. GPR35 is a therapeutic target in inflammatory diseases of the lower gut. In colonic organoids from a human GPR35a-expressing transgenic mouse line, the GPR35 inverse agonist CID-2745687 increased barrier permeability substantially, indicating that constitutive receptor activity contributes to maintaining epithelial barrier integrity. High constitutive activity of GPR35 was also observed in both HT-29 and HEPG2 cells that express GPR35 endogenously. Mechanistic investigations in recombinant in vitro systems revealed that the constitutive activity of GPR35a was biased and not equivalent across signaling pathways. Hence, no constitutive interactions of the receptor with arrestin-adaptor proteins or activation of Gα_o-containing G protein heterotrimers were detected while, even at low GPR35a expression levels, substantial constitutive activation of heterotrimers containing either Gα₁₂ or Gα₁₃ was observed. Similar biased constitutive activity was observed for the human GPR35b isoform. The extent of constitutive and agonist-mediated activity was dependent on receptor expression level. At high receptor levels, constitutive activation of Gα₁₂ or Gα₁₃ masked any agonist-induced effects while low expression levels with low constitutive activity allowed measurement of agonist-induced responses. These results highlight roles, selectivity, and the extent of constitutive activity of GPR35 in cells and tissues that express this receptor endogenously and highlight the contribution of its constitutive activity to maintaining the colonic epithelial barrier, potentially limiting the development of inflammatory bowel diseases.