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

Jamet S, Bubnell J, Pfister P, Tomoiaga D, Rogers ME, Feinstein P - PLoS ONE (2015)

Bottom Line: 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.We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Hunter College, CUNY, New York, New York, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Mutational analysis for the β2 adrenergic receptor.(A) Workflow schematic of the mutational analysis for the β2 adrenergic receptor. First step was to reduce the primary sequence of the mouse β2AR (mβ2AR) to that of the non-trafficking odorant receptor M71. Then we asked what is the C-terminus minimal requirement for trafficking and activity by analyzing Ct truncations series as well as Ct full deletion. Furthermore we analyzed the effects of mutating a putative retention signal that might be revealed by a Ct deletion. We also compared the phenotype of mouse and human β2AR activity mutants to M71. Finally, we analyzed the behavior of M71/mβ2AR chimera. (B) The 2D topology of mouse β2AR was generated using the online software Topo2 (see Material & Methods). Plasma membrane is symbolized by two horizontal lines with the extracellular compartment on top and intracellular compartment on the bottom. Putative transmembrane domains are located in between those two lines. Different shapes and colors are used to symbolize the residues targeted for mutations. N-linked glycosylation sites are indicated in black boxes and their N in diamonds.
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pone.0141696.g001: Mutational analysis for the β2 adrenergic receptor.(A) Workflow schematic of the mutational analysis for the β2 adrenergic receptor. First step was to reduce the primary sequence of the mouse β2AR (mβ2AR) to that of the non-trafficking odorant receptor M71. Then we asked what is the C-terminus minimal requirement for trafficking and activity by analyzing Ct truncations series as well as Ct full deletion. Furthermore we analyzed the effects of mutating a putative retention signal that might be revealed by a Ct deletion. We also compared the phenotype of mouse and human β2AR activity mutants to M71. Finally, we analyzed the behavior of M71/mβ2AR chimera. (B) The 2D topology of mouse β2AR was generated using the online software Topo2 (see Material & Methods). Plasma membrane is symbolized by two horizontal lines with the extracellular compartment on top and intracellular compartment on the bottom. Putative transmembrane domains are located in between those two lines. Different shapes and colors are used to symbolize the residues targeted for mutations. N-linked glycosylation sites are indicated in black boxes and their N in diamonds.

Mentions: We performed a mutational analysis to test the role of N-linked glycosylation of the β2AR amino-terminus (Nt) on its trafficking capacity; we modified mβ2AR so that its primary sequence is analogous to a non-trafficking GPCR, the M71 OR, to test if the length of the Nt and/or Ct were factors involved in trafficking; we defined the role of the minimal Ct and chimeras with M71 OR on proper β2AR trafficking and activity. Finally, we analyzed known mβ2AR activity mutants on their in vitro trafficking defects. Over 30 mutants were analyzed. The workflow of this mutagenesis is summarized in Fig 1A.


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

Jamet S, Bubnell J, Pfister P, Tomoiaga D, Rogers ME, Feinstein P - PLoS ONE (2015)

Mutational analysis for the β2 adrenergic receptor.(A) Workflow schematic of the mutational analysis for the β2 adrenergic receptor. First step was to reduce the primary sequence of the mouse β2AR (mβ2AR) to that of the non-trafficking odorant receptor M71. Then we asked what is the C-terminus minimal requirement for trafficking and activity by analyzing Ct truncations series as well as Ct full deletion. Furthermore we analyzed the effects of mutating a putative retention signal that might be revealed by a Ct deletion. We also compared the phenotype of mouse and human β2AR activity mutants to M71. Finally, we analyzed the behavior of M71/mβ2AR chimera. (B) The 2D topology of mouse β2AR was generated using the online software Topo2 (see Material & Methods). Plasma membrane is symbolized by two horizontal lines with the extracellular compartment on top and intracellular compartment on the bottom. Putative transmembrane domains are located in between those two lines. Different shapes and colors are used to symbolize the residues targeted for mutations. N-linked glycosylation sites are indicated in black boxes and their N in diamonds.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4626089&req=5

pone.0141696.g001: Mutational analysis for the β2 adrenergic receptor.(A) Workflow schematic of the mutational analysis for the β2 adrenergic receptor. First step was to reduce the primary sequence of the mouse β2AR (mβ2AR) to that of the non-trafficking odorant receptor M71. Then we asked what is the C-terminus minimal requirement for trafficking and activity by analyzing Ct truncations series as well as Ct full deletion. Furthermore we analyzed the effects of mutating a putative retention signal that might be revealed by a Ct deletion. We also compared the phenotype of mouse and human β2AR activity mutants to M71. Finally, we analyzed the behavior of M71/mβ2AR chimera. (B) The 2D topology of mouse β2AR was generated using the online software Topo2 (see Material & Methods). Plasma membrane is symbolized by two horizontal lines with the extracellular compartment on top and intracellular compartment on the bottom. Putative transmembrane domains are located in between those two lines. Different shapes and colors are used to symbolize the residues targeted for mutations. N-linked glycosylation sites are indicated in black boxes and their N in diamonds.
Mentions: We performed a mutational analysis to test the role of N-linked glycosylation of the β2AR amino-terminus (Nt) on its trafficking capacity; we modified mβ2AR so that its primary sequence is analogous to a non-trafficking GPCR, the M71 OR, to test if the length of the Nt and/or Ct were factors involved in trafficking; we defined the role of the minimal Ct and chimeras with M71 OR on proper β2AR trafficking and activity. Finally, we analyzed known mβ2AR activity mutants on their in vitro trafficking defects. Over 30 mutants were analyzed. The workflow of this mutagenesis is summarized in Fig 1A.

Bottom Line: 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.We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Hunter College, CUNY, New York, New York, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus