The subject of this thesis is the identification of the interactions that occur between transmembrane (TM) domains of G-protein coupled receptors (GPCRs), with particular emphasis on their roles in the folding, assembly, stability and oligomerization. GPCRs are integral membrane proteins characterized by seven TM helices that mediate a plethora of cellular signals across the cell plasma membrane. They modulate many physiological processes and are linked to numerous diseases but little is know about the determinants of their structures, folding, assembly, activation mechanisms and oligomeric states. GPCRs are also difficult to express and purify, which makes their structural characterization challenging. Therefore, a need exists for detailed structural studies to improve our understanding of the GPCR family. One approach to circumvent these difficulties and gain insight into the folding and assembly processes of integral membrane proteins is to study peptides corresponding to the TM domains. In this thesis, I studied the folding and assembly of the human adenosine A2A receptor (A2AR) as a representative example of human GPCRs.
We first studied in detail the folding and self-assembly properties of the TM5 domain peptide of A2AR. To characterize this peptide in membrane-like environments, we used circular dichroism (CD), gel electrophoresis, fluorescence and Forster resonance energy transfer (FRET) and analytical ultracentrifugation. We found that TM5 peptide forms dimeric structures when inserted in SDS micelles and DMPC vesicles. Then, we performed site-directed mutagenesis on the full-length A2AR to identify residues susceptible to affect receptor oligomerization. We established that mutation at position M193 in the fifth TM helix disrupts the dimers formed by A2AR. Finally, the oligomerization state of mutant receptors in living cells was tested using CFP- and YFP-tagged receptors in saturation binding assays, confocal microscopy and fluorescence spectroscopy experiments. Our results showed that the M193A mutation affects receptor trafficking to the cells plasma membrane by preventing A2AR dimer formation, confirming our in vitro results. These findings strongly indicate that the TM5 domain of A2AR is part of the contact interface between two receptors, and that M193 plays a major role in the oligomerization process.
We also studied the interactions between synthetic peptides corresponding to the seven TM domains of A2AR in order to test the hypothesis that interactions between TM domains are required for proper insertion and folding. First, we monitored the interactions between pairs of TM peptides inserted in membrane-mimetic environments by CD spectroscopy. We established that some pairs interact productively to increase helical content, suggesting that interaction between adjacent TMs can help helix formation and assembly. Then, we investigated the TM5/TM6 as a representative pair in further detail using variants of the TM6 peptide and different spectroscopic methods such as CD and fluorescence spectroscopy, and FRET. This allowed us to develop and verify our methodology. Finally, we performed a systematic analysis of the interactions between all seven helices of A2AR mixed in pair wise combination using FRET. We found that the α-helical content of the peptide plays a role in the folding and the interaction. We also identified a subset of specific interactions between TM domains, which we believe are related to the stability and/or the folding of the receptor. Overall, we were able to postulate from our results a model for the folding and assembly of the full-length A2AR in the membrane during its biogenesis through the translocon machinery.
In these studies, we showed that we could robustly identify and characterize interactions between TM domains of a GPCR. This is the first time that such a comprehensive study has been accomplished using peptides corresponding to TM domains of a membrane protein. The ability to identify crucial inter-helical contacts is a valuable tool in the understanding of the stability and the assembly of these important and challenging proteins.