Impact of the DRY Motif and the Missing "Ionic Lock" on Constitutive Activity and G-Protein Coupling of the Human Histamine H-4 Receptor

Abstract

It is assumed that many G protein-coupled receptors (GPCRs) are restrained in an inactive state by the "ionic lock," an interaction between an arginine in transmembrane domain (TM) 3 (R3.50) and a negatively charged residue in TM6 (D/E6.30). In the human histamine H-4 receptor (hH(4)R), alanine is present in position 6.30. To elucidate whether this mutation causes the high constitutive activity of hH(4)R, we aimed to reconstitute the ionic lock by constructing the A6.30E mutant. The role of R3.50 was investigated by generating hH(4)R-R3.50A. Both mutants were expressed alone or together with G alpha(i2) and G beta(1)gamma(2) in Sf9 cells and characterized in GTPase, S-35-labeled guanosine 5'-[ gamma-thio] triphosphate binding, and high-affinity agonist binding assays. Unexpectedly, compared with hH(4)R, hH(4)R-A6.30E showed only nonsignificant reduction of constitutive activity and G protein-coupling efficiency. The K-D of [H-3] histamine was unaltered. By contrast, hH(4)R-R3.50A did not stimulate G proteins. Thioperamide affinity at hH(4)R-R3.50A was increased by 300 to 400%, whereas histamine affinity was reduced by approximately 50%. A model of the active hH(4)R state in complex with the G alpha(i2) C terminus was compared with the crystal structures of turkey beta(1) and human beta(2) adrenoceptors. We conclude that 1) constitutive activity of hH(4)R is facilitated by the salt bridge D5.69-R6.31 rather than by the missing ionic lock, 2) Y3.60 may form alternative locks in active and inactive GPCR states, 3) R3.50 is crucial for hH(4)R-G protein coupling, and 4) hH(4)R-R3.50A represents an inactive state with increased inverse agonist and reduced agonist affinity. Thus, the ionic lock, although stabilizing the inactive rhodopsin state, is not generally important for all class A GPCRs.