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Constitutively Active Mutants of the Histamine H₁ Receptor Suggest a Conserved Hydrophobic Asparagine-Cage That Constrains the Activation of Class A G Protein-Coupled Receptors

Leiden/Amsterdam Center for Drug Research, Department of Medicinal Chemistry, Vrije Universiteit Amsterdam, Amsterdam, the Netherlands (R.A.B., A.J., K.S., H.T., and R.L.); ACADIA Pharmaceuticals Inc., San Diego, California (R.A.B., U.H., M.R.B., and D.M.W.); Department of Pharmacology, University of California at San Diego, San Diego, California (M.R.B.); Department of Neurosciences, University of California at San Diego, San Diego, California (D.M.W.); Department of Psychiatry, University of California at San Diego, San Diego, California (D.M.W.); and Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain (L.P.)

The aim of this study was to create and characterize constitutively active mutant (CAM) histamine H₁ receptors (H₁R) using random mutagenesis methods to further investigate the activation process of the rhodopsin-like family of G protein-coupled receptors (GPCRs). This approach identified position 6.40 in TM 6 as a "hot spot" because mutation of Ile6.40⁴²⁰ either to Glu, Gly, Ala, Arg, Lys, or Ser resulted in highly active CAM H₁Rs, for which almost no histamine-induced receptor activation response could be detected. The highly conserved hydrophobic amino acid at position 6.40 defines, in a computational model of the H₁R, the asparagine cage motif that restrains the side chain of Asn7.49 of the NPxxY motif toward transmembrane domain (TM 6) in the inactive state of the receptor. Mutation of the asparagine cage into Ala or Gly, removing the interfering bulky constraints, increases the constitutive activity of the receptor. The fact that the Ile6.40⁴²⁰Arg/Lys/Glu mutant receptors are highly active CAM H₁Rs leads us to suggest that a positively charged residue, presumably the highly conserved Arg3.50 from the DRY motif, interacts in a direct or an indirect (through other side chains or/and internal water molecules) manner with the acidic Asp2.50··Asn7.49 pair for receptor activation.

Address correspondence to: Dr. R. Leurs, Leiden/Amsterdam Center for Drug Research, Department of Medicinal Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. E-mail: r.leurs{at}few.vu.nl