The largest class of cell surface receptors in mammalian genomes is the superfamily of G protein-coupled receptors (GPCRs) which are activated by a wide range of extracellular responses such as hormones, pheromones, odorants, and neurotransmitters. Drugs which have therapeutic effects on a wide range of diseases are act on GPCRs. In contrast to traditional idea, it is recently getting accepted that G-protein coupled receptors can form homo- and hetero-dimers and this interaction could have important role on maturation, internalization, function or/and pharmacology. Bimolecular fluorescence complementation technique (BiFC) / is an innovative approach based on the reassembly of protein fragments which directly report interactions. In our study we implemented this technique for detecting and visualizing the GPCR interactions in yeast cells. The enhanced green fluorescent protein (EGFP) fractionated into two fragments at genetic level which does not possess fluorescent function. The target proteins which are going to be tested in terms of interaction are modified with the non-functional fragments, to produce the fusion proteins. The interaction between two target proteins, in this study Ste2p receptors which are alpha pheromone receptors from Saccharomyces cerevisiae, enable the fragments to come in a close proximity and reassemble. After reassembly, EGFP regains its fluorescent function which provides a direct read-out for the detection of interaction. Further studies are required to determine subcellular localization of the interaction. Moreover, by using the fusion protein partners constructed in this study, effects of agonist/antagonist binding and post-translational modifications such as glycosylation and phosphorylation can be examined. Apart from all, optimized conditions for BiFC technique will guide for revealing new protein-protein interactions.

The G protein-coupled receptor (GPCR) family is composed of hundreds of members and is expressed in eukaryotes. Each GPCR has seven transmembrane domains and is in charge of sensing changes from the environment, transducing signals, and activating a series of biological responses. The signal transduction pathway of the receptor starts from sensing outside signal and then activates G proteins. This signaling requires a tight control for activation without which impaired cellular function leads to pathology. We have used the pheromone alpha-factor receptor (Ste2p) of the yeast Saccharomyces cerevisiae as a model system to understand ligand binding, receptor activation, and G protein interaction. One method we have used to study ligand binding is to incorporate the photo-reactive crosslinker p-benzoyl-L-phenylalanine (Bpa) into Ste2p to capture alpha-factor. This powerful tool requires the incorporation of Bpa, an unnatural amino acid, into Ste2p by a special genetic manipulation designed in the lab of Peter Schulz (Scripps Institute) and adapted by our lab for Ste2p. Another method to study ligand binding that we have adapted for use in our system is to incorporate a chemical crosslinker [3,4-dihydroxylphenylacetyl (DHPA)] into alpha-factor for periodate-mediated crosslinking to Ste2p. The interacting domain between alpha-factor and transmembrane domain 2 to 3 of Ste2p was identified after DOPAC crosslinking, cyanogen bromide digestion and MALDI-TOF mass spectrometry. After ligand binding, signal transduction is mediated by the interaction of activated Ste2p with its G protein (Gpa1p). We studied this interaction by replacing natural residues in the intracellular loop 3 of Ste2p and C-terminal end of Gpa1p with cysteine and then determining disulfide crosslinking between Ste2p and Gpa1p. Some residues were found to be in close proximity and displayed different interacting patterns due to conformational changes of the receptor upon ligand binding. The information we gathered here allows us to understand more about the physical interactions of alpha-factor, Ste2p, and Gpa1p and provides us insights about the initiation and activation of the signal transduction pathway of a peptide ligand receptor.