Primary structure of C-terminal functional sites in ovine rhodopsin

Findlay, John B. C.; Brett, M.; Pappin, Darryl

doi:10.1038/293314a0

Cited by 64 publications

(43 citation statements)

References 25 publications

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“…1). Similarly, the 1 1-cis-retinal chromophore of the visual pigments is covalently attached to Lys296 located in transmembrane helix VII of bovine rhodopsin Findlay et al, 1981), and is believed to interact with residues on the core-facing surface of one or more helices (Applebury & Hargrave, 1986). It has been speculated that, by analogy, the seven a-helices of G protein-coupled receptors may also form a ligand-binding pocket Venter et al, 1989 .…”

Section: The Search For Features Common To G Protein-coupled Receptormentioning

confidence: 99%

In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors

Savarese

Fraser

1992

Biochemical Journal

462

236

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Section: The Search For Features Common To G Protein-coupled Receptormentioning

confidence: 99%

In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors

Savarese

Fraser

1992

Biochemical Journal

462

236

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“…An early hypothesis for rhodopsin was that these transmembrane domains assemble into an annular structure to form the retinal-binding pocket. Indeed, the hydrocarbon chain of retinal was shown to be covalently attached to Lys296 in the middle ofTMS VII (72)(73)(74)(75), and to lie close to the center of the transmembrane domains, parallel to the plane of the lipid bilayer (76).…”

Section: Determinants For Ligand Bindingmentioning

confidence: 99%

“…An acidic residue (Asp1l 3) in TMS III of the (3rAR is similarly conserved among all 7-TMS receptors that bind biogenic amines , and it was proposed that such an acidic residue could form an ion pair with the protonated amine moiety of the ligand (86) [72][73][74][75]. By itself, the presence of a positive charge in the hydrophobic transmembrane environment might be energetically unfavorable; however, a Glu residue that could serve as a stabilizing coun terion has been identified by site-directed mutagenesis (87,88).…”

Section: Determinants For Ligand Bindingmentioning

confidence: 99%

Model Systems For The Study Of Seven-Transmembrane-Segment Receptors

Dohlman¹

1991

Annual Review of Biochemistry

197

203

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“…And finally, evidence has accumulated over the last decade that the transmembrane portion of these receptors is also involved in ligand bind-ing. In bovine rhodopsin, for instance, the light-absorbing retinal chromophore is located within the transmembrane region, covalently linked to residue Lys296 in helix VII (Findlay et al, 1981). In the case of small ligands like the biogenic amines, sitc-directed mutagenesis experiments have located the ligandbinding pocket within the transmembrane region of the receptors which seems to be composed of several amino acid residues belonging to different helices (Savarese and Fraser, 1992).…”

mentioning

confidence: 99%

Site‐Directed Mutagenesis of Conserved Charged Residues in the Helical Region of the Human C5a Receptor

Raffetseder

Méry

et al. 1996

European Journal of Biochemistry

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(Rcccived 25 Julyi2 October 1995) ~ E3B 95 122XilThe human CSa receptor (C5aR) belongs to the family of G-protein-coupled receptors with seven transmembrane helices. This part of the molecule is thought to contain part of the ligand-binding pocket, specifically to bind the C-terminal Arg of human CSa. Guided by sequence similarity and molecular modelling studies, several residues including polar (Asnll9, Thr168, Gln259) as well as all conserved charged amino acids in the upper transmembrane region of the CSaR (Asp37, Asp82, Arg175, Arg206, Asp282) were exchanged by site-directed mutagenesis. Receptor mutants were transiently expressed in COS cells and analyzed for altered binding behaviour and/or localization at the cell surface by immunofluorescence. For all residues, suitable mutants could be found that exhibited wild-type affinity towards the ligand, providing evidence against a major contribution of these residues to high-affinity ligand binding. Some mutants, however, exhibited a complete (Asp282--+Ala) or partial loss of ligandbinding capacity (Argl75+Ala, Arg206+Gln) despite adequate expression levels on the cell surface. This phenotype was further analyzed in the [Gln206]CSaR mutant: quantitative flow cytometric analysis of epitope-tagged receptor derivatives in 293 cells confirmed an equal level of wild-type and mutant C5aR on the cell surface. Competitive binding curves revealed the presence of only a small population ( < l o % ) of high-affinity sites ( K p 2 nM), which was functionally active at 20 nM in the heterologous Xencyus oocyte expression system after coexpression of Ga-16. The number of high-affinity sites of wild-type and [Gln206]C5aR in 293 cells could be up-regulated by coexpression of Gia-2 and downregulated by GTP[ yS ]-mediated uncoupling of the G-protein receptor interaction in membrane preparations. These findings are compatible with a model in which the Arg206 residue located in the upper third of transmembrane helix V determines high-affinity binding in the human CSaR by affecting the intracellular G-protein coupling.Keywords: C5a receptor; receptor structure; guanine-nucleotide-binding-regulatory-protein coupling; site-directed mutagenesis.Human complement factor C5a (C5a) is a 74-amino-acid glycopeptide which is generated by proteolytic cleavage from the fifth component of complement. Binding of the CSa peptide to its receptor triggers major cell activation and degranulation events, such as chemotaxis of polymorphonuclear cells, induction of the oxidative burst, histamine and interleukin release rrom monocytes. C5a is therefore thought to be an important inflammation-promoting agent. It interacts with the C5a receptor (CSaR) found in the membrane of polymorphonuclear cells, monocytes, macrophages and the histiocytic U937 and HL-60 cell lines (for a recent review, see KBhl and Bitter-Suermann, 1993, and the literature cited therein).

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Primary structure of C-terminal functional sites in ovine rhodopsin

Cited by 64 publications

References 25 publications

In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors

In vitro mutagenesis and the search for structure-function relationships among G protein-coupled receptors

Model Systems For The Study Of Seven-Transmembrane-Segment Receptors

Site‐Directed Mutagenesis of Conserved Charged Residues in the Helical Region of the Human C5a Receptor

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