The Role of Transmembrane Helix 5 in Agonist Binding to the Human H3 Receptor

Abstract

We have used alanine scanning mutagenesis to identify residues in transmembrane domain 5 of the histamine H3 receptor that are important for agonist binding. All of the mutants generated were functionally expressed as demonstrated by their ability to bind [125 I]iodoproxyfan with comparable affinity to the wild-type receptor and their ability to inhibit forskolin-stimulated cAMP formation when activated by histamine. Many mutations produced small changes in the potency of histamine, but the most pronounced reduction in potency and affinity of the agonists, histamine, R-␣-methylhistamine, imetit, and impentamine, was seen with mutation of glutamate 206. Our modeling suggests that this residue plays a key role in ligand binding by interacting with the imidazole ring of histamine. Interestingly, L199A greatly reduced agonist potency in functional assays but had only minor effects on agonist affinity, implicating a role for this residue in the mechanism of receptor activation. We also studied the functional effects of the mutations by linking the receptor to calcium signaling using a chimeric G protein. A comparison of the two functional assays demonstrated contrasting effects on agonist activity. Histamine, imetit, and impentamine were full agonists in the cAMP assay, but imetit exhibited only partial agonist activity through the chimeric G protein. Furthermore, impentamine, another potent agonist in the cAMP assay, was only able to activate the E206A mutant in the calcium assay despite being inactive at the wild-type receptor. These observations suggest that the agonist receptor complexes formed by these three different H3 agonists are not conformationally equivalent.

Generation of a homology model of the human histamine H3 receptor for ligand docking and pharmacophore-based screening

“…Several rotamers were considered for each residue, for which no definite placement could be obtained by applying the SCWRL algorithm and which was likely participating in the binding site. The approximate position of antagonists in the hH 3 R binding pocket was thereby known from mutational studies, which showed that D3.32 and E5.46 were the major sites of interaction [22]. During docking FUB836 into all alternative binding sites, a distance constraint between the piperidyl-nitrogen of FUB836 and D3.32 was applied.…”

“…Another residue showing a similar ''unstable'' behaviour was F5.47, which adopted a rotamer pointing into the binding pocket during the simulation of uncomplexed hH 3 R models. For the F5.47A variant a significant drop in potency was observed suggesting that this residue was involved in upholding the receptor structure or in receptor activation [22]. In simulations of antagonist/hH 3 R complexes the conformational switch of F5.47 towards the inside of the binding pocket was inhibited due to the presence of the ligand.…”

“…In the complexes studied, the imidazole moiety of antagonists was-in analogy with the imidazole moiety of histamine-assumed to interact with E5.46 2 . In 2002, Uveges and co-workers studied the natural agonist histamine in an hH 3 R homology model [22]. Histamine was manually placed such as to contact E5.46 with its imidazole moiety and D3.32 with its primary amine functionality.…”

Structure-based discovery and binding site analysis of histamine receptor ligands

“…A bovine rhodopsin based model of the H3R was used to investigate the role of TM5 residues in ligand binding [36]. Mutation of Glu206(5.46) showed reduction of affinity and potency of several agonists, such as histamine, R-α-methylhistamine, imetit and impentamine.…”

Molecular modeling and mutagenesis of gap junction channels

“…The role of individual residues in transmembrane helices has been traditionally addressed by scanning mutagenesis (generally Cys-or Ala-scanning mutagenesis), where the TM residues are substituted, one at a time, and the functional and/or structural consequences of the substitutions are investigated (Kaback et al, 2001;Frillingos et al, 1998;Lu et al, 2001;Uveges et al, 2002). In the case of one of the better studied membrane proteins, the lactose permease of E. coli (12 TMs), it has been established that only 6 of its 417 amino acids are essential for function and cannot be replaced with any other residue (Kaback et al, 2001;Abramson et al, 2003).…”