Analysis of Missense Variants in the Human Histamine Receptor Family Reveals Increased Constitutive Activity of E4106.30×30K Variant in the Histamine H1 Receptor

Abstract

The Exome Aggregation Consortium has collected the protein-encoding DNA sequences of almost 61,000 unrelated humans. Analysis of this dataset for G protein-coupled receptor (GPCR) proteins (available at GPCRdb) revealed a total of 463 naturally occurring genetic missense variations in the histamine receptor family. In this research, we have analyzed the distribution of these missense variations in the four histamine receptor subtypes concerning structural segments and sites important for GPCR function. Four missense variants R1273.52×52H, R13934.57×57H, R4096.29×29H, and E4106.30×30 K, were selected for the histamine H1 receptor (H1R) that were hypothesized to affect receptor activity by interfering with the interaction pattern of the highly conserved D(E)RY motif, the so-called ionic lock. The E4106.30×30 K missense variant displays higher constitutive activity in G protein signaling as compared to wild-type H1R, whereas the opposite was observed for R1273.52×52H, R13934.57×57H, and R4096.29×29H. The E4106.30×30 K missense variant displays a higher affinity for the endogenous agonist histamine than wild-type H1R, whereas antagonist affinity was not affected. These data support the hypothesis that the E4106.30×30 K mutation shifts the equilibrium towards active conformations. The study of these selected missense variants gives additional insight into the structural basis of H1R activation and, moreover, highlights that missense variants can result in pharmacologically different behavior as compared to wild-type receptors and should consequently be considered in the drug discovery process.

Pharmacological characterization of seven human histamine H3receptor isoforms

“…Importantly, aspartic or glutamic acid residues at position 6.30x30 have been identified to form an interhelical ionic lock with the highly conserved arginine at position 3.50x50 in the DRY motif in the intracellular end of TM3 to stabilize inactive receptor conformations (Palczewski et al, 2000). Site-directed mutagenesis or genetic variants of aspartic or glutamic acid at 6.30x30 have been found to disrupt this ionic lock in many different GPCRs such as for example histamine H 1 receptor (Ma et al, 2021), β 2 -adrenergic receptor (Ballesteros et al, 2001), serotonin 5HT 2A receptor (Shapiro et al, 2002), resulting in increased constitutively signaling and associated changes ligand binding affinities. Hence, the absence of an ionic lock between TM3 and TM6 is hypothesized to cause the higher constitutive activity of H 3 R-365 and H 3 R-373.…”

“…have been found to disrupt this ionic lock in many different GPCRs such as for example histamine H1 receptor (Ma et al, 2021), b2-adrenergic receptor (Ballesteros et al, 2001), serotonin 5HT2A receptor (Shapiro et al, 2002), resulting in increased constitutively signaling and associated changes ligand binding affinities. Hence, the absence of an ionic lock between TM3 and TM6 is hypothesized to cause the higher constitutive activity of H3R-365 and H3R-373.…”

The World of GPCR dimers – Mapping dopamine receptor D2 homodimers in different activation states and configuration arrangements

“…Arg 1.59 has been reported to be relevant for G protein binding, particularly in the interaction between Gα s and Gα i1 for the β 1 -adrenoceptor [148] . According to Wang et al, residues at position 3.52, Thr 3.52 for D 2 R, would only be relevant for G protein binding if they were hydrophobic [149] , [150] . Overall, residues on TM4, such as Arg 4.40 and Arg 4.41, are important for homo- and heterodimers comprising the TM4-TM5 interface and can be pathogenic when mutated.…”

BRET-Based Biosensors to Measure Agonist Efficacies in Histamine H1 Receptor-Mediated G Protein Activation, Signaling and Interactions with GRKs and β-Arrestins

“…Human embryonic kidney 293T (HEK293T) cells (ATCC; Manassas, VA, USA) were maintained in DMEM supplemented with 10% FBS, 50 IU mL −1 penicillin and 50 mg mL −1 streptomycin in a humidified incubator at 37 °C with 5% CO 2 . HEK293T cells (2 × 10 6 cells/dish) were seeded in a 10 cm dish and transiently transfected the next day using the PEI method, as previously described [ 63 ]. To this end, plasmid DNA was mixed with 20 µg L-PEI (25 kDa) in 0.25 mL NaCl solution (150 mM) and incubated for 30 min at 22 °C before adding dropwise to a 10 cm dish with HEK293T cells.…”