RT Journal Article SR Electronic T1 Impact of the DRY Motif and the Missing “Ionic Lock” on Constitutive Activity and G-Protein Coupling of the Human Histamine H4 Receptor JF Journal of Pharmacology and Experimental Therapeutics JO J Pharmacol Exp Ther FD American Society for Pharmacology and Experimental Therapeutics SP 382 OP 392 DO 10.1124/jpet.109.163220 VO 333 IS 2 A1 Erich H. Schneider A1 David Schnell A1 Andrea Strasser A1 Stefan Dove A1 Roland Seifert YR 2010 UL http://jpet.aspetjournals.org/content/333/2/382.abstract AB 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 H4 receptor (hH4R), alanine is present in position 6.30. To elucidate whether this mutation causes the high constitutive activity of hH4R, we aimed to reconstitute the ionic lock by constructing the A6.30E mutant. The role of R3.50 was investigated by generating hH4R-R3.50A. Both mutants were expressed alone or together with Gαi2 and Gβ1γ2 in Sf9 cells and characterized in GTPase, 35S-labeled guanosine 5′-[γ-thio]triphosphate binding, and high-affinity agonist binding assays. Unexpectedly, compared with hH4R, hH4R-A6.30E showed only nonsignificant reduction of constitutive activity and G protein-coupling efficiency. The KD of [3H]histamine was unaltered. By contrast, hH4R-R3.50A did not stimulate G proteins. Thioperamide affinity at hH4R-R3.50A was increased by 300 to 400%, whereas histamine affinity was reduced by approximately 50%. A model of the active hH4R state in complex with the Gαi2 C terminus was compared with the crystal structures of turkey β1 and human β2 adrenoceptors. We conclude that 1) constitutive activity of hH4R 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 hH4R–G protein coupling, and 4) hH4R-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. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics