Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor

Abstract

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Selective phosphorylation of threonine residues defines GPR84–arrestin interactions of biased ligands

“…Mass spectrometry was performed at the University of Leicester Proteomics Facility. Samples were analyzed as described (40). Liquid chromatography/tandem mass spectrometry (LC-MS/MS) was carried out using an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific).…”

Isoforms of GPR35 have distinct extracellular N-termini that allosterically modify receptor-transducer coupling and mediate intracellular pathway bias

“…Besides coupling to heterotrimeric G proteins, GPR35 isoforms interact with -arrestin2, a process that depends on agonist-promoted phosphorylation of a species-conserved serine/threonine cluster in the receptor C-terminus (29). Following the profiling of G protein activation and signaling, we next investigated isoform-specific GPR35--arrestin interaction and receptor internalization.…”

How Arrestins and GRKs Regulate the Function of Long Chain Fatty Acid Receptors

“…However, the use of selective small molecule GRK inhibitors, such as compound 101 to block GRK 2/3 [ 117 ] and compound 18 to block GRK5/6 [ 118 ], siRNA/shRNA methods [ 116 , 119 ] or CRISPR/Cas9 approaches targeting only a specific subset of relevant GRKs [ 107 , 120 ], is beginning to unravel the mysteries of these topics. In addition, the use of phospho-site-specific antibodies [ 16 , 121 , 122 ] and mass spectrometry analysis of the sites of regulated phosphorylation [ 16 , 96 , 122 , 123 ] are also providing valuable insights. For example, Marsango et al (2022) identified a crucial pair of threonine residues in the medium chain fatty acid receptor GPR84 that are only phosphorylated in response to receptor activation, and this occurs, at least in HEK293 cells, via GRK2/3 [ 16 ].…”

“…This regulation defines efficient interactions with arrestins and allows the separation of G protein-biased and more balanced GPR84 agonists [ 16 ]. Similarly, Divorty et al (2022) reported phospho-site-specific antisera that act as activation state-specific biomarkers for the orphan metabolite receptor GPR35 [ 122 ]. Here, pre-treatment with the GRK2/3 blocker compound 101 significantly decreased the agonist-induced phosphorylation of human, and particularly mouse, orthologues of GPR35, as detected by these antisera.…”