Our collaboration on receptors with professor Luigi F Agnati from the University of Modena, resulted in two publications. Agnati LF, Marcoli M, Maura G, Woods A, Guidolin D. The brain as a hyper network: the key role of neural networks as main producers of the integrated brain actions especially via the broadcasted neuroconnectomics Journal of Neural Transmission. 125, 883897 (2018) and Cervetto C, Venturini A, Passalacqua M, Guidolin D, Genedani S, Fuxe K, Borroto-Escuela DO, Cortelli P, Woods A, Maura G, Marcoli M and Agnati LF. A2A-D2 receptor-receptor interaction in modulates of glliotransmitter release from striatal astrocyte processes., J. Neurochem. 140, 268-279. (2017). Our work targets the receptors involved in addiction, to explain how their structure dictates how they interact with other receptors and what makes these interactions specific. Our data suggest that heteromer formation, mainly involves linear motifs found in disordered regions of proteins. Local disorder imparts plasticity to linear motifs. Many molecular recognition of proteins occur between short linear segments, known as LMs. Interaction of short continuous epitopes are not constrained by sequence and have the advantage of resulting in interactions with micromolar affinities which suites transient, reversible complexes such as receptor heteromers. Electrostatic Interactions between epitopes of the GPCR involved, is the Key step in driving heteromer formation forward. The first step in heteromerization, involves phosphorylating the Ser/Thr, in an epitope containing a casein kinase 1/2 (CK1/2)-consensus site. Our data suggests that dopaminergic neurotransmission, through cAMP dependent Protein kinase A (PKA) slows down heteromerization. The negative charge, acquired by the phosphorylation of a Ser/Thr in a PKA consensus site in the Arg rich epitope affects the activity of the receptors involved in heteromerization by causing allosteric conformational changes, due to the repulsive effect generated by the negatively charged phosphate. In addition to modulating heteromerization, it affects the stability of the heteromers interactions and heir binding affinity. So here we have an instance where phosphorylation is not just an on/off switch, instead by weakening the noncovalent bond, heteromerization acts like a rheostat that controls the stability of the heteromer through activation or inhibition of adenylate cyclase by the neurotransmitter Dopamine depending on which Dopamine receptor it docks at. Complex molecular and cellular mechanisms regulate G protein-coupled receptors (GPCRs). It is suggested that proteins intrinsically disordered regions (IDRs) are to play a role in GPCRsintra and extracellular regions plasticity, due to their potential for post-translational modification and interaction with other proteins. These regions are defined as lacking a stable three-dimensional (3D) structure. They are rich in hydrophilic cand charged, amino acids and are capable to assume different conformations which allow them to interact with multiple partners. In this study we analyzed 75 GPCR involved in synaptic transmission using computational tools for sequence-based prediction of IDRs within a protein. We also evaluated putative ligand-binding motifs using receptor sequences. The disorder analysis indicated that neurotransmitter GPCRs have a significant amount of disorder in their N-terminus, third intracellular loop (3IL) and C-terminus. About 31%, 39% and 53% of human GPCR involved in synaptic transmission are disordered in these regions. Thirty-three percent of receptors show at least one predicted PEST motif, this being statistically greater than the estimate for the rest of human GPCRs. About 90% of the receptors had at least one putative site for dimerization in their 3IL or C-terminus. ELM instances sampled in these domains were 14-3-3, SH3, SH2 and PDZ motifs. In conclusion, the increased flexibility observed in GPCRs, added to the enrichment of linear motifs, PEST and heteromerization sites, maybe critical for the nervous systems functional plasticity. Certain amino acid residues and posttranslational modifications play an important role in the formation of noncovalent complexes (NCXs) by electrostatic interactions. Electrospray ionization mass spectrometry(ESI-MS) is the most widely used MS technique for the study of NCXs, due to its softer ionization process and compatibility with the solution phase of NCX mixtures. In order to locate the site where interactions are forming in the NCXs involving phosphopeptides and adjacent arginines, tandem mass spectrometry studies using collision-induced dissociation (CID) and electron transfer dissociation (ETD) were performed on NCXs at different charge states. CID fragmentation revealed two dissociation pathways:one in which the electrostatic interaction is disrupted and another in which the covalent bond attaching the phosphate group to the amino acid residue is cleaved, while the electrostatic interaction is maintained. ETD and sequential ETD/ETD, and CID/ETD allow the determination of the NCX interaction site. These results confirmed the involvement of the phosphorylated amino acid and at least two adjacent arginines as the binding site.
