GPR35: Study of Class a GPCR Sequence Divergences using Conformational Memories

Abstract

GPR35, a recently deorphanized Class A G-protein coupled receptor, shows prominent expression in immune and gastrointestinal tissues (Wang et al. JBC, 2006), with additional expression in pancreatic islets, skeletal muscle, and lung (Horikawa et al. Nat. Genet. 2000), brain and spinal cord. An endogenous ligand of GPR35, kynurenic acid, is one of the major metabolites of the kynurenine pathway; a pathway in which the main route of tryptophan catabolism has been associated with important physiological roles in the brain. Compared with prototypical Class A GPCRs such as the beta-2-adrenergic receptor (β2AR; Cherezov et al. Science 2007), GPR35 has several major sequence differences: (1) in TMH4, GPR35 lacks the Pro at 4.60; (2) in TMH2 there is a Pro shift from 2.59 to 2.58; and, (3) in TMH5, there is an additional Pro at position 5.43. In order to study the consequences of these sequence divergences on the GPR35 structure, we used the Monte Carlo/simulated annealing program, Conformational Memories (Whitnell et. al, J. Comput. Chem. 2007). Each helix was built using the standard phi and psi angles for TMHs, −62.9°/-41.6° (Ballesteroes et al. Meth. Neuro. 1995). CM calculations revealed that the extracellular end of GPR35 TMH4 diverges from that of the β2AR, as the bend, wobble angle and face shift was (26.2°,40.0°,48.0°) compared to the average of 105 CM structures for GPR35 TMH4 (18.04° ± 4.95°, −123.39°± 58°,1.63° ± 11.83°). The GPR35 one residue N-terminal TMH2 proline shift created a face shift of 53.04°± 27.21° compared to 96.8° for the β2AR TMH2. The additional proline in GPR35 TMH5 resulted in a bend and face shift of (12.76° ± 4.74°, 47.43°± 22.74°) vs. (6.0°, 80.1°) for the β2AR. [Support: NIH RO1 DA023204 (MEA) and KO5 DA021358 (PHR)]