Abstract
G-protein coupled receptors (GPCRs) are a class of
integral membrane receptor proteins that are characterized by a
signature seven-transmembrane (7-TM) configuration. The
a-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR
that, upon binding of a peptide ligand, transduces a signal to
initiate a cascade of events leading to the mating of haploid yeast
cells. This study summarizes the application of affinity purification
and of matrix-assisted laser-desorption ionization time-of-flight
(MALDI-TOF) experiments using biotinylated photoactivatable
a-factor analogs. Affinity purification and enrichment of
biotinylated peptides by monomeric avidin beads resulted in mass
spectrometric detection of specific signals corresponding to crosslinked
fragments of Ste2p. Data obtained from cyanogen bromide
(CNBr) fragments of receptor cross-linked to an a-factor analog
with the photoaffinity group p-benzoyl-L-phenylalanine on
position 1 were in agreement with the previous results reported by
our laboratory suggesting the cross-linking between position 1 of
a-factor and a region of Ste2p covering residues 251–294.