Premium
Cross‐Linking of a DOPA‐Containing Peptide Ligand into its G Protein‐Coupled Receptor
Author(s) -
Umanah George E.,
Son Cagdas D,
Ding FaXiang,
Naider Fred,
Becker Jeffrey M
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.880.8
Subject(s) - chemistry , g protein coupled receptor , peptide , residue (chemistry) , biochemistry , cysteine , receptor , stereochemistry , enzyme
The interaction between a 3,4‐dihydroxylphenylalanine (DOPA) labeled analog of the tridecapeptide α‐factor and Ste2p, a Saccharomyces cerevisiae model G protein‐coupled receptor (GPCR), has been analyzed by periodate‐mediated cross‐linking. Chemically synthesized α‐factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine 7 [Lys 7 (Biotin),Nle 12 ,DOPA 13 ]α ‐factor (Bio‐DOPA‐α ‐factor) was used for cross‐linking into Ste2p. The biological activity of Bio‐DOPA‐α ‐factor was about one‐third that of native α‐factor and exhibited about a ten‐fold lower binding affinity. Bio‐DOPA‐α ‐factor cross‐linked into Ste2p, and cross‐linking was inhibited by native α‐factor. 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‐α ‐factor‐Ste2p cross‐linked peptides. Mass‐spectrometric analyses of the cross‐linked fragments showed that Bio‐DOPA‐α ‐factor reacted with the Phe 55 ‐Met 69 region of Ste2p. Cross‐linking of Bio‐DOPA‐α ‐factor was reduced by 80% using a cysteine‐less Ste2p (Cys59Ser). These results suggest an interaction between position thirteen of α‐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 α‐factor. Supported by NIH GM‐22086 and GM‐22087 1