title: A fluorescent alpha-factor analogue exhibits multiple steps on binding to its G protein coupled receptor in yeast.
abstract: The yeast alpha-factor receptor encoded by the STE2 gene is a member of the extended family of G protein coupled receptors (GPCRs) involved in a wide variety of signal transduction pathways. We report here the use of a fluorescent alpha-factor analogue [K(7)(NBD), Nle(12)] alpha-factor (Lys(7) (7-nitrobenz-2-oxa-1,3-diazol-4-yl), norleucine(12) alpha-factor) in conjunction with flow cytometry and fluorescence microscopy to study binding of ligand to the receptor. Internalization of the fluorescent ligand following receptor binding can be monitored by fluorescence microscopy. The use of flow cytometry to detect binding of the fluorescent ligand to intact yeast cells provides a sensitive and reproducible assay that can be conducted at low cell densities and is relatively insensitive to fluorescence of unbound and nonspecifically bound ligand. Using this assay, we determined that some receptor alleles expressed in cells lacking the G protein alpha subunit exhibit a higher equilibrium binding affinity for ligand than the same alleles expressed in isogenic cells containing the normal complement of G protein subunits. On the basis of time-dependent changes in the intensity and shape of the emission spectrum of [K(7)(NBD),Nle(12)] alpha-factor during binding, we infer that the ligand associates with receptors via a two-step process involving an initial interaction that places the fluorophore in a hydrophobic environment, followed by a conversion to a state in which the fluorophore moves to a more polar environment.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, P.O. Box 712, Rochester, New York 14642, USA.
authors.forename: Anshika
authors.initials: A
authors.lastname: Bajaj
authors.forename: Andjelka
authors.initials: A
authors.lastname: Celić
authors.forename: Fa-Xiang
authors.initials: FX
authors.lastname: Ding
authors.forename: Fred
authors.initials: F
authors.lastname: Naider
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Becker
authors.forename: Mark E
authors.initials: ME
authors.lastname: Dumont
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism*; D018628:Gene Dosage; D008024:Ligands; D008239:Lysine / Q000378:metabolism; D000072235:Mating Factor; D008856:Microscopy, Fluorescence; D009646:Norleucine / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D010455:Peptides; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000072235:Mating Factor; D009646:Norleucine; D009327:4-Chloro-7-nitrobenzofurazan; D008239:Lysine