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Graph analysis of ?2 adrenergic receptor structures: a "social network" of GPCR residues.

ABSTRACT: PURPOSE:G protein-coupled receptors (GPCRs) are a superfamily of membrane proteins of vast pharmaceutical interest. Here, we describe a graph theory-based analysis of the structure of the ?2 adrenergic receptor (?2 AR), a prototypical GPCR. In particular, we illustrate the network of direct and indirect interactions that link each amino acid residue to any other residue of the receptor. METHODS:Networks of interconnected amino acid residues in proteins are analogous to social networks of interconnected people. Hence, they can be studied through the same analysis tools typically employed to analyze social networks - or networks in general - to reveal patterns of connectivity, influential members, and dynamicity. We focused on the analysis of closeness-centrality, which is a measure of the overall connectivity distance of the member of a network to all other members. RESULTS:The residues endowed with the highest closeness-centrality are located in the middle of the seven transmembrane domains (TMs). In particular, they are mostly located in the middle of TM2, TM3, TM6 or TM7, while fewer of them are located in the middle of TM1, TM4 or TM5. At the cytosolic end of TM6, the centrality detected for the active structure is markedly lower than that detected for the corresponding residues in the inactive structures. Moreover, several residues acquire centrality when the structures are analyzed in the presence of ligands. Strikingly, there is little overlap between the residues that acquire centrality in the presence of the ligand in the blocker-bound structures and the agonist-bound structures. CONCLUSIONS:Our results reflect the fact that the receptor resembles a bow tie, with a rather tight knot of closely interconnected residues and two ends that fan out in two opposite directions: one toward the extracellular space, which hosts the ligand binding cavity, and one toward the cytosol, which hosts the G protein binding cavity. Moreover, they underscore how interaction network is by the conformational rearrangements concomitant with the activation of the receptor and by the presence of agonists or blockers.

SUBMITTER: Sheftel S

PROVIDER: S-EPMC4230308 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Graph analysis of β2 adrenergic receptor structures: a "social network" of GPCR residues.

Sheftel Samuel S Muratore Kathryn E KE Black Michael M Costanzi Stefano S

In silico pharmacology 20131205

<h4>Purpose</h4>G protein-coupled receptors (GPCRs) are a superfamily of membrane proteins of vast pharmaceutical interest. Here, we describe a graph theory-based analysis of the structure of the β2 adrenergic receptor (β2 AR), a prototypical GPCR. In particular, we illustrate the network of direct and indirect interactions that link each amino acid residue to any other residue of the receptor.<h4>Methods</h4>Networks of interconnected amino acid residues in proteins are analogous to social netw ...[more]

PMID: 25505660

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Project description:Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse β2-adrenergic-receptor (mβ2AR), robustly traffics to the plasma membrane. We set out to characterize mβ2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of mβ2AR using a Green Fluorescent Protein-tagged mβ2AR (mβ2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known mβ2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of mβ2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of mβ2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three mβ2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower mβ2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs.

Dataset Information

Graph analysis of ?2 adrenergic receptor structures: a "social network" of GPCR residues.

Publications

Graph analysis of β2 adrenergic receptor structures: a "social network" of GPCR residues.

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