Previous studies revealed pharmacological differences between human and guinea pig histamine H 2 receptors (H 2 Rs) with respect to the interaction with guanidine-type agonists. Because H 2 R species variants are structurally very similar, comparative studies are suited to relate different properties of H 2 R species isoforms to few molecular determinants. Therefore, we systematically compared H 2 Rs of human (h), guinea pig (gp), rat (r), and canine (c). Fusion proteins of hH 2 R, gpH 2 R, rH 2 R, and cH 2 R, respectively, and the short splice variant of G s␣ , G s␣S , were expressed in Sf9 insect cells. In the membrane steady-state GTPase activity assay, cH 2 R-G s␣S but neither gpH 2 R-G s␣S nor rH 2 R-G s␣S showed the hallmarks of increased constitutive activity compared with hH 2 R-G s␣S , i.e., increased efficacies of partial agonists, increased potencies of agonists with the extent of potency increase being correlated with the corresponding efficacies at hH 2 R-G s␣S , increased inverse agonist efficacies, and decreased potencies of antagonists. Furthermore, in membranes expressing nonfused H 2 Rs without or together with mammalian G s␣S or H 2 R-G s␣ fusion proteins, the highest basal and GTP-dependent increases in adenylyl cyclase activity were observed for cH 2 R. An example of ligand selectivity is given by metiamide, acting as an inverse agonist at hH 2 R-G s␣S , gpH 2 R-G s␣S , and rH 2 R-G s␣S in the GTPase assay in contrast to being a weak partial agonist with decreased potency at cH 2 R-G s␣S . In conclusion, the cH 2 R exhibits increased constitutive activity compared with hH 2 R, gpH 2 R, and rH 2 R, and there is evidence for ligand-specific conformations in H 2 R species isoforms.The histamine H 2 receptor (H 2 R) species isoforms of canine (Gantz et al., 1991b), human (Gantz et al., 1991a), rat (Ruat et al., 1991), and guinea pig (Traiffort et al., 1995) were cloned. The four H 2 R species isoforms are closely related to each other, as is reflected by an overall amino acid sequence identity of more than 80%. The highest conservation exists within the seven ␣-helical transmembrane (TM) domains (sequence identity of more than 90%), whereas the N-terminal domain together with the extracellular end of TM1 and the C terminus are the least conserved regions (Fig. 1).Despite this high degree of structural similarity, N-[3-(1H-imidazol-4-yl)propyl]guanidines such as compounds 8 to 10 (Fig. 2) differentially activate guinea pig (gpH 2 R) and human (hH 2 R) H 2 receptors. In a membrane steady-state GTPase activity assay using fusion proteins of H 2 R and the short splice variant of G s␣ , G s␣S , such H 2 R-selective agonists are considerably more potent and efficacious at gpH 2 R-G s␣S than at hH 2 R-G s␣S (Kelley et al., 2001). By contrast, the small H 2 R agonists histamine (1, HA), dimaprit (2, DIM), amthamine (3, AMT), and betahistine (4, BET) are unselective between these species. Recently, a novel class of N G -acylated imida- Article, publication date, and citation information can be fo...

Residues in the second extracellular loop (e2) play a role in ligand binding in certain aminergic G protein coupled receptors (GPCRs). N-[3-(1H-Imidazol-4-yl)propyl)]guanidines and N (G)-acylated derivatives are more efficacious and potent agonists at fusion proteins of the guinea pig histamine H(2) receptor and the short splice variant of G(salpha), G(salphaS) (gpH(2)R-G(salphaS)) than at the human isoform (hH(2)R-G(salphaS)). To elucidate the structural basis for this species-selectivity, we generated a mutant hH(2)R-G(salphaS) fusion protein with the four e2 residues differing in both species isoforms mutated into the gpH(2)R sequence, and a reverse mutant of the gpH(2)R-G(salphaS) with the corresponding mutations into the human species. In a steady-state GTPase activity assay, efficacies and potencies of guanidine-type agonists were similar at mutant and wild-type receptors indicating that e2 does not contribute to the species-selectivity. In several class 1 GPCRs, amino acids in the vicinity of a highly conserved cysteine in e2 participate in ligand binding. A three-dimensional homology model of the hH(2)R predicted Lys-173 and Lys-175, adjacent to Cys-174 in e2, to be in close proximity to the binding pocket of guanidine-type agonists. To elucidate the putative role of both residues for interactions with the agonists, two hH(2)R-G(salphaS) fusion proteins, with single-point mutations of Lys-173-->Ala-173 and Lys-175-->Ala-175 respectively, were generated. With these mutants, the efficacies and potencies of small and bulky H(2)R agonists did not significantly change. However, increases in GTPase activity upon agonist stimulation were reduced, suggesting an impact of both residues on the efficiency of receptor coupling to G(salphaS). In conclusion, none of the point mutations generated within this study substantially altered the efficacies and potencies of guanidine-type agonists relative to the wild-type receptors, suggesting that these residues do not directly face the H(2)R guanidine-binding pocket. Thus, agonist binding to residues in e2 is relevant for some but not all aminergic GPCRs.

In a steady-state GTPase activity assay, N- [3-(1H-imidazol-4-yl)propyl)]guanidines and N G -acylated derivatives are more potent and efficacious at fusion proteins of guinea pig (gpH 2 R-G s␣S ) than human (hH 2 R-G s␣S ) histamine H 2 receptor, coupled to the short splice variant of G s␣ , G s␣S . Whereas Ala-271 (hH 2 R) and Asp-271 (gpH 2 R) in transmembrane domain 7 were identified to determine the potency differences of guanidine-type agonists, the molecular basis for the efficacy differences remains to be elucidated. A homology model of the gpH 2 R suggested that an H-bond between Tyr-17 and Asp-271 stabilizes an active receptor conformation of the gpH 2 R. In the present study, we generated a mutant hH 2 R-G s␣S with Cys-173 Tyr-17/Ala-2713 Asp-271 exchanges (hH 2 R3gpH 2 R) that exhibited an enhanced level of constitutive GTPase activity and adenylyl cyclase activity compared with wild-type hH 2 R-G s␣S and gpH 2 R-G s␣S . Potencies and efficacies of guanidines and N G -acylguanidines were increased at this mutant receptor compared with hH 2 R-G s␣S , but they were still lower than at gpH 2 R-G s␣S , suggesting that aside from Tyr-17 and Asp-271 additional amino acids contribute to the distinct pharmacological profiles of both species isoforms. Another hH 2 R-G s␣S mutant with a Cys-173 Tyr-17 exchange showed inefficient coupling to G s␣S as revealed by reduced agonist-stimulated GTPase and basal adenylyl cyclase activities. Collectively, our present pharmacological study confirms the existence of an H-bond between Tyr-17 and Asp-271 favoring the stabilization of an active receptor conformation. Distinct potencies and efficacies of agonists and inverse agonists further support the concept of ligand-specific conformations in wild-type and mutant H 2 R-G s␣S fusion proteins.The histamine H 2 receptor (H 2 R) is a biogenic amine receptor that belongs to the class A of the family of GPCRs. After stimulation by histamine (HA; Fig. 1, 1), the H 2 R couples to G s proteins to activate adenylyl cyclase (AC). H 2 Rs mediate regulation of gastric acid secretion in parietal cells, cardiac contractility, and myeloid cell differentiation (Del Valle and Gantz, 1997).N- propyl]guanidines are the most potent agonists at the H 2 R known so far (up to 400 times more active than HA at the guinea pig right atrium), and they are possibly useful as positive inotropic drugs for the treatment of severe congestive heart failure, as agents inducing cell differentiation in acute myelogenous leukemia, and as anti-inflammatory drugs (Dove et al., 2004). Guanidines are less potent and efficient agonists at the Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.120519.ABBREVIATIONS: H 2 R, histamine H 2 receptor; GPCR, G protein-coupled receptor; HA, histamine; AC, adenylyl cyclase; G s␣ , ␣-subunit of the G s protein that mediates adenylyl cyclase activation; G s␣S , short splice variant of the G s protein G s␣ ; gpH 2 R, guinea pig histamine H 2 receptor; ...

The phototropin from Chlamydomonas reinhardtii is a 120 kDa blue light receptor that plays a key role in gametogenesis of this green alga. It comprises two light-sensing domains termed LOV1 and LOV2 (light oxygen and voltage) and a serine/threonine kinase domain. The post-translationally incorporated chromophore is flavin mononucleotide (FMN). Upon absorption of blue light, LOV domains undergo a photocycle that activates a Ser/Thr kinase. The mechanism of this activation is still unknown. We studied the oligomerization of the recombinant LOV1 domain (amino acids 16-133) of C. reinhardtii by means of UV/Vis spectroscopy, size-exclusion chromatography (SEC), and chemical cross-linking with glutardialdehyde. The thermal back-reaction of LOV1 from the signaling state to the dark state as monitored by UV/Vis spectroscopy after an intensive blue light pulse could not be explained by a monoexponential model, although the spectra did not indicate the presence of an additional species. Therefore, we investigated the quaternary structure of the LOV1 domain by size-exclusion chromatography in the dark. This revealed an equilibrium between dimers and higher oligomers (M(W)>200 kDa) under native conditions. No monomers were detected by SEC. However, by analysis of the equilibrium by cross-linking of the protein with glutardialdehyde and subsequent SDS-PAGE, monomers and dimers were identified. Exposure of LOV1 to blue light resulted in a decrease in the monomer/dimer ratio, followed by re-equilibration in the dark. Calculation of the solvent-accessible surface area and the Conolly surfaces of the LOV1 dimers present in the crystal structure support the experimental observation that no mononomers are detected in the native state. A model is presented that accounts for a blue-light-driven change in the quaternary structure of the LOV1 domain and gives hints to the molecular basis of light activation and regulation in LOV-containing proteins.