Photoaffinity labeling of rhodopsin and bacteriorhodopsin

Nakanishi, Koji; Zhang, Hongzhi; Lerro, Keith A.; Takekuma, Shin‐ichi; Yamamoto, Toshihiro; Lien, Thoai Hung; Sastry, Lakshmi; Baek, Du-Jong; Moquin-Pattey, Carole; Boehm, Marcus F.; Derguini, Fadila; Gawinowicz, Mary Ann

doi:10.1016/0301-4622(95)00010-u

Cited by 39 publications

(22 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Both of these sites, as is the case for the ligand binding site of FPR, map to transmembrane regions near the extracellular face of these receptors. The ligand binding sites on FPR, the ␤ adrenergic receptor, and the muscarinic receptor, all map to a region very similar to that found for the retinal binding site of rhodopsin (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). Therefore, we conclude that the binding sites for many different types of GPCR ligands appear to reside in a very similar region of GPCR within the transmembrane region near the extracellular face of the membrane.…”

Section: Fluoresceinated Adducts From Both Wild Type and Fprexpressinmentioning

confidence: 64%

Identification of a Ligand Binding Site in the Human Neutrophil Formyl Peptide Receptor Using a Site-specific Fluorescent Photoaffinity Label and Mass Spectrometry

Mills

Miettinen

Barnidge

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

The phagocyte chemotactic receptors, including the formyl peptide receptor (FPR), 1 the lipoxin A 4 receptor, the C5a receptor, the platelet-activating factor receptor, and the interleukin-8 receptor are involved in inflammation and are all members of the G protein-coupled receptor (GPCR) superfamily. Among the most studied in this inflammatory receptor family is neutrophil FPR (1). FPR binds N-formyl peptides, such as formyl-Met-Leu-Phe (fMLF), with nanomolar affinity (2). Such N-formyl peptides are indicators of the presence of bacteria (3) or damage to host cell mitochondria (4,5). Binding of N-formyl peptides to FPR thus provides phagocytes with signals for infection or injury and results in activation of chemotaxis and other host defensive processes including lysosomal enzyme secretion, stimulation of production of inflammatory mediators, and generation of superoxide.The effects of amino acid substitutions and modifications of fMLF peptides on binding to FPR and activation have been studied extensively (6 -8). The formyl group, the methionine at position 1, and phenylalanine at position 3 have been shown to be necessary for high affinity binding. Decarboxylation of the C-terminal phenylalanine markedly reduces activity, but esters or amides of this residue or peptides with C-terminal amino acid additions exhibit similar activity to the tripeptide with the free acid. None of the fMLF functional groups have been shown to be absolutely essential for activity but rather they appear to individually contribute to the overall free energy of binding.Chemical and photoaffinity cross-linking of fMLF analogs to FPR has been achieved by a number of groups (9 -12). However, none of these studies have identified the site of labeling. The residue in the second position of N-formylated peptides appears to be the most tolerant of modification (6, 13) and both of the flanking residues are critical for high affinity binding (14); so the second residue was chosen to accommodate the photoreactive amino acid benzoylphenylalanine (Bpa). Bpa is chemically stable in the absence of photoexcitation, can be directly introduced into peptide ligands by solid phase peptide synthesis, and has been photocross-linked into several peptide receptors (15). Here we report that a fluorescent photoaffinity analog of fMLF, formyl-Met-p-benzoyl-L-phenylalanine-PheTyr-Lys-⑀-N-fluorescein (fMBpaFYK-fl), efficiently photocrosslinks to FPR residues 83-85. Derivatization of these residues in FPR by Bpa supports recent site-directed mutagenesis stud-* This work was supported in part by National Science Foundation EPSCoR Grant RII-891879 (to E. A. D.), a Grant from the Pittsburgh Supercomputing Centers through the National Institutes of Health resource Grant 2p41RR06009, a grant from the Rocky Mountain Chapter of the Arthritis Foundation and the Harmon Foundation (to J. S. M.), a grant from the Rocky Mountain Chapter of the Arthritis Foundation (to H. M. M.), and Public Health Service Grants 1RO1A40108-01 and RO122735 (to A. J. J.). The costs of publicati...

show abstract

Section: Fluoresceinated Adducts From Both Wild Type and Fprexpressinmentioning

confidence: 64%

Identification of a Ligand Binding Site in the Human Neutrophil Formyl Peptide Receptor Using a Site-specific Fluorescent Photoaffinity Label and Mass Spectrometry

Mills

Miettinen

Barnidge

et al. 1998

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…In rhodopsin the region of helix F (TMS VI) containing the conserved proline residue (proline-267) is located near the ␤-ionone ring of retinal when the chromophore exists in the cis isomer (Nakanishi et al, 1995), which maintains the inactive conformation of rhodopsin. When rhodopsin is activated by light, the environment of tryptophan-265 in helix F changes (Lin and Sakmar, 1996) and a rigid-body motion of helix F relative to helix C (TMS III) appears to occur .…”

Section: Gpcr Activation Mechanismsmentioning

confidence: 99%

Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

Stefan

Overton

Blumer

1998

MBoC

View full text Add to dashboard Cite

We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate agonist-induced structural changes leading to G protein activation. Constitutive signaling by mutant receptors is suppressed upon coexpression with wild-type, but not G protein coupling-defective, receptors. Wild-type receptors may therefore sequester a limiting pool of G proteins; this apparent "precoupling" of receptors and G proteins could facilitate signal production at sites where cell surface projections form during mating partner discrimination. Finally, rather than being expressed mainly at the cell surface, constitutively active pheromone receptors accumulate in post-endoplasmic reticulum compartments. This is in contrast to other defective membrane proteins, which apparently are targeted by default to the vacuole. We suggest that the quality-control mechanism that retains receptors in post-endoplasmic reticulum compartments may normally allow wild-type receptors to fold into their native, fully inactive conformations before reaching the cell surface. This may ensure that receptors do not trigger a response in the absence of agonist.

show abstract

“…The GPCR superfamily is particularly important because these proteins are the targets of a large fraction of pharmaceuticals and genomic analysis indicates that perhaps 5% of the proteins coded by the human genome are GPCRs (11). Functional interactions of membrane proteins in signaling or adhesion pathways are often studied via photo cross-linking of peptides or radioligands to the proteins (12)(13)(14). Detailed structural information can be provided by MS analysis of the cross-linking sites (13,15).…”

mentioning

confidence: 99%

Mass Spectrometric Analysis of Cyanogen Bromide Fragments of Integral Membrane Proteins at the Picomole Level: Application to Rhodopsin

Kraft

Mills

Dratz

2001

Analytical Biochemistry

View full text Add to dashboard Cite

Photoaffinity labeling of rhodopsin and bacteriorhodopsin

Cited by 39 publications

References 38 publications

Identification of a Ligand Binding Site in the Human Neutrophil Formyl Peptide Receptor Using a Site-specific Fluorescent Photoaffinity Label and Mass Spectrometry

Identification of a Ligand Binding Site in the Human Neutrophil Formyl Peptide Receptor Using a Site-specific Fluorescent Photoaffinity Label and Mass Spectrometry

Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

Mass Spectrometric Analysis of Cyanogen Bromide Fragments of Integral Membrane Proteins at the Picomole Level: Application to Rhodopsin

Contact Info

Product

Resources

About