Structural and functional analysis of the canine histamine H2 receptor by site-directed mutagenesis: N-glycosylation is not vital for its action

Fukushima, Yasushi; Oka, Yoshitomo; Saitoh, Toshihito; Katagiri, Hideki; Asano, Tomohiko; Matsuhashi, Nobuyuki; Takata, Kuniaki; Breda, Eric van; Yazaki, Yoshio; Sugano, Kentaro

doi:10.1042/bj3100553

Cited by 66 publications

(32 citation statements)

References 24 publications

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“…Further, site-directed mutagenesis at two of three consensus sites of the histamine H2 receptor show that lack of glycosylation does not alter the receptor's ability to bind ligand, nor does it affect cAMP activation and intracellular calcium accumulation. Immunostaining and binding experiments localized the glycosylationdefective receptors to the plasma membrane, implying that N-glycosylation is not required for intracellular targeting of the H2 receptor (41).…”

Section: Discussionmentioning

confidence: 99%

Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

Tansky

Pothoulakis²,

Leeman³

2007

Proc. Natl. Acad. Sci. U.S.A.

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The neurokinin 1 receptor (NK1R), a G protein-coupled receptor involved in diverse functions including pain and inflammation, has two putative N-linked glycosylation sites, Asn-14 and Asn-18. We studied the role of N-linked glycosylation in the functioning of the NK1R by constructing three receptor mutants: two single mutants (Asn 3 Gln-14 and Asn 3 Gln-18) and a double mutant, lacking both glycosylation sites. Using a lentiviral transfection system, the mutants were stably transfected into NCM 460 cells, a nontransformed human colonic epithelial cell line. We observed that the magnitude of glycosylation as estimated by changes in gel migration depends on the number of glycosylation sites available, with the wild-type receptor containing the greatest amount of glycosylation. All mutant receptors were able to bind to substance P and neurokinin A ligand with similar affinities; however, the double mutant, nonglycosylated NK1R showed only half the B max of the wild-type NK1R. In terms of receptor function, the ablation of both N-linked glycosylation sites did not have a profound effect on the receptors' abilities to activate the MAP kinase families (p42/p44, JNK, and p38), but did affect SP-induced IL-8 secretion. All mutants were able to internalize, but the kinetics of internalization of the double mutant receptor was more rapid, when compared with wild-type NK1R. Therefore, glycosylation of NK1R may stabilize the receptor in the plasma membrane. These results contribute to the ongoing elucidation of the role of glycosylation in G proteincoupled receptors and the study of the neurokinin receptors in particular.G protein-coupled receptor ͉ substance P

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Section: Discussionmentioning

confidence: 99%

Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

Tansky

Pothoulakis²,

Leeman³

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…H 2 R species isoforms presumably exhibit similar glycosylation patterns, because the putative N-glycosylation sites for the H 2 R, Asn-4, and Asn-162 are fully conserved within their sequences ( Fig. 1) (Fukushima et al, 1995). However, only rH 2 R and hH 2 R migrated as the expected bands for monomeric GPCRs (Fig.…”

Section: Immunological Detection Of Recombinant Proteins In Sf9mentioning

confidence: 97%

Constitutive Activity and Ligand Selectivity of Human, Guinea Pig, Rat, and Canine Histamine H₂Receptors

Preuss

Ghorai²,

Kraus³

et al. 2007

J Pharmacol Exp Ther

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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...

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“…For example, the lack of N-glycosylation may lead to virtually complete intracellular retention, as has been reported for human vasoactive intestinal peptide 1 and rat AT 1a angiotensin II receptors (6,7). Frequently, however, the expression levels merely decrease, as happens for rat prostaglandin E 2 and human AT 1 receptors (8,9), or the non-N-glycosylated receptors are fully functional and properly localized at the plasma membrane, as is the case for canine histamine H2 and porcine m2 muscarinic acetylcholine receptors (10,11). Furthermore, these attributes are not necessarily dependent on N-glycosylation as such but on a particular N-glycan, in a specific position.…”

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confidence: 90%

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

Markkanen

Petäjä-Repo

2008

Journal of Biological Chemistry

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A great majority of G protein-coupled receptors are modified by N-glycosylation, but the functional significance of this modification for receptor folding and intracellular transport has remained elusive. Here we studied these phenomena by mutating the two N-terminal N-glycosylation sites (Asn 18 and Asn 33 ) of the human ␦-opioid receptor, and expressing the mutants from the same chromosomal integration site in stably transfected inducible HEK293 cells. Both N-glycosylation sites were used, and their abolishment decreased the steady-state level of receptors at the cell surface. However, pulse-chase labeling, cell surface biotinylation, and immunofluorescence microscopy revealed that this was not because of intracellular accumulation. Instead, the non-N-glycosylated receptors were exported from the endoplasmic reticulum with enhanced kinetics. The results also revealed differences in the significance of the individual N-glycans, as the one attached to Asn 33 was found to be more important for endoplasmic reticulum retention of the receptor. The non-N-glycosylated receptors did not show gross functional impairment, but flow cytometry revealed that a fraction of them was incapable of ligand binding at the cell surface. In addition, the receptors that were devoid of N-glycans showed accelerated turnover and internalization and were targeted for lysosomal degradation. The results accentuate the importance of protein conformation-based screening before export from the endoplasmic reticulum, and demonstrate how the system is compromised when N-glycosylation is disrupted. We conclude that N-glycosylation of the ␦-opioid receptor is needed to maintain the expression of fully functional and stable receptor molecules at the cell surface.

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Structural and functional analysis of the canine histamine H2 receptor by site-directed mutagenesis: N-glycosylation is not vital for its action

Cited by 66 publications

References 24 publications

Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

Constitutive Activity and Ligand Selectivity of Human, Guinea Pig, Rat, and Canine Histamine H₂Receptors

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

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Structural and functional analysis of the canine histamine H2 receptor by site-directed mutagenesis: N-glycosylation is not vital for its action

Cited by 66 publications

References 24 publications

Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

Functional consequences of alteration of N-linked glycosylation sites on the neurokinin 1 receptor

Constitutive Activity and Ligand Selectivity of Human, Guinea Pig, Rat, and Canine Histamine H2Receptors

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

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Constitutive Activity and Ligand Selectivity of Human, Guinea Pig, Rat, and Canine Histamine H₂Receptors