Dataset Information

In Vitro Mutational Analysis of the ?2 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 ?2-adrenergic-receptor (m?2AR), robustly traffics to the plasma membrane. We set out to characterize m?2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of m?2AR using a Green Fluorescent Protein-tagged m?2AR (m?2AR::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 m?2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of m?2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of m?2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three m?2AR 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 m?2AR 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.

SUBMITTER: Jamet S

PROVIDER: S-EPMC4626089 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Publications

In Vitro Mutational Analysis of the β2 Adrenergic Receptor, an In Vivo Surrogate Odorant Receptor.

Jamet Sophie S Bubnell Jaclyn J Pfister Patrick P Tomoiaga Delia D Rogers Matthew E ME Feinstein Paul P

PloS one 20151029 10

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 Prot ...[more]

PMID: 26513247

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Project description:We performed an extensive mutational analysis of the canonical mouse odorant receptor (OR) M71 to determine the properties of ORs that inhibit plasma membrane trafficking in heterologous expression systems. We employed the use of the M71::GFP fusion protein to directly assess plasma membrane localization and functionality of M71 in heterologous cells in vitro or in olfactory sensory neurons (OSNs) in vivo. OSN expression of M71::GFP show only small differences in activity compared to untagged M71. However, M71::GFP could not traffic to the plasma membrane even in the presence of proposed accessory proteins RTP1S or m?2AR. To ask if ORs contain an internal "kill sequence", we mutated ~15 of the most highly conserved OR specific amino acids not found amongst the trafficking non-OR GPCR superfamily; none of these mutants rescued trafficking. Addition of various amino terminal signal sequences or different glycosylation motifs all failed to produce trafficking. The addition of the amino and carboxy terminal domains of m?2AR or the mutation Y289A in the highly conserved GPCR motif NPxxY does not rescue plasma membrane trafficking. The failure of targeted mutagenesis on rescuing plasma membrane localization in heterologous cells suggests that OR trafficking deficits may not be attributable to conserved collinear motifs, but rather the overall amino acid composition of the OR family. Thus, we performed an in silico analysis comparing the OR and other amine receptor superfamilies. We find that ORs contain fewer charged residues and more hydrophobic residues distributed throughout the protein and a conserved overall amino acid composition. From our analysis, we surmise that it may be difficult to traffic ORs at high levels to the cell surface in vitro, without making significant amino acid modifications. Finally, we observed specific increases in methionine and histidine residues as well as a marked decrease in tryptophan residues, suggesting that these changes provide ORs with special characteristics needed for them to function in olfactory neurons.

Dataset Information

In Vitro Mutational Analysis of the ?2 Adrenergic Receptor, an In Vivo Surrogate Odorant Receptor.

Publications

In Vitro Mutational Analysis of the β2 Adrenergic Receptor, an In Vivo Surrogate Odorant Receptor.

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