The interaction between a 3,4-dihydroxylphenylalanine (DOPA) labeled analog of the tridecapeptide α-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 α-factor with DOPA substituting for tyrosine at position 13 and biotin tagged onto lysine 7 ([Lys 7 (BioACA),-Nle 12 ,DOPA 13 ]α-factor; Bio-DOPA-α-factor) was used for crosslinking into Ste2p. The biological activity of Bio-DOPA-α-factor was about one-third that of native α-factor as determined by growth arrest assay and exhibited about a ten-fold lower binding affinity to Ste2p. Bio-DOPA-α-factor cross-linked into Ste2p as demonstrated by western blot analysis using a Neutravidin-HRP conjugate to detect Bio-DOPA-α-factor. Cross-linking was inhibited by excess native α-factor and an α-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-α-factor-Ste2p cross-linked peptides. MALDI-TOF 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 thereby defining a specific contact point between the bound ligand and its receptor.G protein-coupled receptors (GPCRs) are a large family of integral membrane proteins associated with signaling systems present in mammalians, plants, protozoans, fungi, and metazoans (1). Malfunctions of GPCRs contribute to diseases such as Alzheimer's, Parkinson's disease, diabetes, color blindness, asthma, depression, hypertension, stress, cardiovascular, and immune disorders (2,3). All GPCRs share a common structure consisting of seven transmembrane domains (TMDs) connected by three extracellular and three intracellular loops (1,4,5). Upon binding of their ligands, GPCRs undergo conformational changes that transduce NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 March 10. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the signal into the cell by activating a cascade of protein-protein interactions initiated through a heterotrimeric G protein complex (6).Ste2p, the Saccharomyces cerevisiae α-factor pheromone receptor, has been studied as a model for peptide-responsive GPCRs (7,8). Though Ste2p does not share recognizable sequence similarity with mammalian GPCRs or even with Ste3p, the a-factor pheromone receptor of yeast, all GPCRs have strong structural and functional similarities (9,10). For example, the packing and interactions between the fifth and sixth tr...

A detailed analysis of the structure of an 18-residue peptide C252A)] in 1,2-dimyristoyl-sn-glycero-phosphocholine bilayers was carried out using solid state NMR and attenuated total reflection Fourier transform infrared spectroscopy. The peptide corresponds to a portion of the 6th transmembrane domain of the α-factor receptor of Saccharomyces cerevisiae. Ten homologs of M6(252-269, C252A) were synthesized in which individual residues were labeled with 15 N. One-and two-dimensional solid state NMR experiments were used to determine the chemical shifts and 1 H-15 N dipolar coupling constants for the 15 N-labeled peptides in oriented dimyristoylphosphatidylcholine bilayers on stacked glass plates. These parameters were used to calculate the structure and orientation of M6(252-269, C252A) in the bilayers. The results indicate that the carboxyl terminal residues (9-14) are αhelical and oriented with an angle of about 8° with respect to the bilayer normal. Independently, an attenuated total reflection Fourier transform infrared spectroscopy analysis on M6(252-269, C252A) in a 1,2-dimyristoyl-sn-glycero-phosphocholine bilayer concluded that the helix tilt angle was about 12.5°. The results on the structure of M6(252-269, C252A) in bilayers are in good agreement with the structure determined in trifluoroethanol/water solutions (B. Arshava et al.