Cross-linking of a DOPA-containing peptide ligand into its G protein-coupled receptor.
The interaction between a 3,4-dihydroxyphenylalanine (DOPA) labeled analogue of the tridecapeptide alpha-factor (W-H-W-L-Q-L-K-P-G-Q-P-M-Y) and Ste2p, a Saccharomyces cerevisiae model G protein-coupled receptor (GPCR), has been analyzed by periodate-mediated cross-linking. Chemically synthesized alpha-factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine(7)([Lys(7)(BioACA),Nle(12),DOPA(13)]alpha-factor; Bio-DOPA-alpha-factor) was used for cross-linking into Ste2p. The biological activity of Bio-DOPA-alpha-factor was about one-third that of native alpha-factor as determined by growth arrest assay and exhibited about a 10-fold lower binding affinity to Ste2p. Bio-DOPA-alpha-factor cross-linked into Ste2p as demonstrated by Western blot analysis using a neutravidin-HRP conjugate to detect Bio-DOPA-alpha-factor. Cross-linking was inhibited by excess native alpha-factor and an alpha-factor antagonist. The Ste2p-ligand complex was purified using a metal ion affinity column, and after cyanogen bromide treatment, avidin affinity purification was used to capture Bio-DOPA-alpha-factor-Ste2p cross-linked peptides. MALDI-TOF spectrometric analyses of the cross-linked fragments showed that Bio-DOPA-alpha-factor reacted with the Phe(55)-Met(69) region of Ste2p. Cross-linking of Bio-DOPA-alpha-factor was reduced by 80% using a cysteine-less Ste2p (Cys59Ser). These results suggest an interaction between position 13 of alpha-factor and residue Cys(59) of Ste2p. This study is the first to report DOPA cross-linking of a peptide hormone to a GPCR and the first to identify a residue-to-residue cross-link between Ste2p and alpha-factor, thereby defining a specific contact point between the bound ligand and its receptor.
Umanah
George K E
GK
Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996, USA.
Son
Cagdas
C
Ding
FaXiang
F
Naider
Fred
F
Becker
Jeffrey M
JM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004295:Dihydroxyphenylalanine / Q000737:chemistry*; D015151:Immunoblotting; D008024:Ligands; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051964:Receptors, Mating Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D003432:Cross-Linking Reagents; D008024:Ligands; D009842:Oligopeptides; D043562:Receptors, G-Protein-Coupled; D051964:Receptors, Mating Factor; C498031:STE2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; D004295:Dihydroxyphenylalanine; D001120:Arginine; D003545:Cysteine