title: In Vitro Mutational Analysis of the β2 Adrenergic Receptor, an In Vivo Surrogate Odorant Receptor.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Hunter College, CUNY, New York, New York, United States of America.
authors.forename: Sophie
authors.initials: S
authors.lastname: Jamet
authors.affiliation: Department of Biological Sciences, Hunter College, CUNY, New York, New York, United States of America.
authors.forename: Jaclyn
authors.initials: J
authors.lastname: Bubnell
authors.affiliation: Corporate Research and Development, Firmenich Inc., Plainsboro, New Jersey, United States of America.
authors.forename: Patrick
authors.initials: P
authors.lastname: Pfister
authors.affiliation: Department of Biological Sciences, Hunter College, CUNY, New York, New York, United States of America.
authors.forename: Delia
authors.initials: D
authors.lastname: Tomoiaga
authors.affiliation: Corporate Research and Development, Firmenich Inc., Plainsboro, New Jersey, United States of America.
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Rogers
authors.affiliation: Department of Biological Sciences, Hunter College, CUNY, New York, New York, United States of America; The Graduate Center Biology Program, CUNY, New York, New York, United States of America; The Graduate Center Behavioral and Cognitive Neuroscience Program, CUNY, New York, New York, United States of America.
authors.forename: Paul
authors.initials: P
authors.lastname: Feinstein
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004252:DNA Mutational Analysis; D015870:Gene Expression; D017930:Genes, Reporter; D006031:Glycosylation; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D051379:Mice; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D021381:Protein Transport; D011554:Pseudopodia / Q000235:genetics / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018035:Receptors, Odorant / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D000318:Adrenergic beta-Agonists; D018343:Receptors, Adrenergic, beta-2; D018035:Receptors, Odorant; D007545:Isoproterenol