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

Abstract

The aim of this study was to create and characterize constitutively active mutant (CAM) histamine H 1 receptors (H 1 R) 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 Ile6.40 420 either to Glu, Gly, Ala, Arg, Lys, or Ser resulted in highly active CAM H 1 Rs, 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 H 1 R, 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 Ile6.40 420 Arg/Lys/Glu mutant receptors are highly active CAM H 1 Rs 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 Asp2.50⅐⅐Asn7.49 pair for receptor activation.

A Structural Insight into the Reorientation of Transmembrane Domains 3 and 5 during Family A G Protein-Coupled Receptor Activation

“…We have shown previously that mutation of the highly conserved hydrophobic side chain at position 6.40 to glutamic acid, glycine, alanine, arginine, lysine, or serine results in highly constitutively active receptors for which almost no additional histamine-induced receptor activation can be detected (Bakker et al, 2008). These substitutions at position 6.40 seem to modify the conformation of the key Asn 7.49 , Tyr 7.53 , and Arg 3.50 side chains, located in the coupling region near the G protein binding site (Bakker et al, 2008). Notably, adding the I 3.40 A mutation, in the trigger region, to the constitutively active I 6.40 S/K mutant receptors decreases constitutive activity and impedes histamine-induced receptor activation (Fig.…”

“…5, bottom left arrow). Disruption of these ionic interactions permits Arg 3.50 to adopt an extended conformation (Bakker et al, 2008;Park et al, 2008), pointing toward the protein core and to interact with the highly conserved Tyr 5.58 and Tyr 7.53 in TMs 5 and 7 . These conformational changes open a cavity between TMs 3, 5, and 6 where the G protein can bind .…”

“…Wildtype, T 3.37 A, and T 3.37 E mutant H 1 Rs were well expressed in COS-7 cells at approximately 10 pmol/mg protein as determined by radioligand binding analysis (Table 1). The function of the WT H 1 R was evaluated in an NF-B reporter-gene assay as reported previously (Jongejan et al, 2005;Bakker et al, 2008), and the activation of NF-B was increased up to 10-fold (E max ϭ 1002 Ϯ 34%) when stimulated with histamine (Fig. 3, A and B).…”

“…We have previously used the H 1 R as a model system for the study of class A GPCR activation (Bakker et al, 2008). The H 1 R belongs to the aminergic subfamily of Class A GPCRs and, like the ␤ 1 -and ␤ 2 -adrenergic receptors, features Thr 3.37 and Ile 3.40 in TM3.…”

“…We combined activating point mutations leading to agonist-independent constitutive activity with the inactivating I 3.40 A/G mutation with the aim to assess their compensatory consequences on receptor function. We have chosen the previously reported S 3.36 T mutation (Jongejan et al, 2005) in the ligand binding site and the I 6.40 K/S mutations (Bakker et al, 2008) near the cytoplasmic site, both leading to a large increase in constitutive activity (see Discussion). The S 3.36 T mutation induces the transition of Trp 6.48 toward TM5 (Jongejan et al, 2005), considered to be the initial stage of the activation process (see below), as observed in the electron microscopy density map of metarhodopsin I (Ruprecht et al, 2004) and in solid-state NMR measurements of metarhodopsin II (Crocker et al, 2006 (Fig.…”