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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.


Reducing mouse β2AR primary sequence to that of the odorant receptor M71.(A) mβ2AR::GFP showing homogenous and sharp edges staining of OP6 cells (observed in 10/10 cells), is included in small isolated inclusions and also locates to filopodia (arrow head in magnified picture). (B) On the contrary, ΔCt➔R328 shows accumulation around the nucleus and does not locate to filopodia (9/10 cells, magnified picture). (C) The mutated final version of mβ2AR that looks like an OR, NtΔ9AA/ΔCt➔Y350/ΔC341, shows similar localization in OP6 cells as WT, including in filopodia (arrow head in magnified picture). (D) In the same way No-NxS shows proper trafficking in OP6 cells. (E) Dose response curves obtained by the degree of isoprenaline-induction in FLIPR experiments, are depicted as normalized relative fluorescent units (RFUs) as a function of Log [isoprenaline] (M, molar concentration). The same color code is used as in F. (F) The average number of GFP-labeled filopodia per cell is showed (bars in graph, mean +s.d.) as well as the number of cells with ≤1 GFP-labeled filopodia (blue diamonds). WT labels 31.8 ±16.2 filopodia per cell, ΔCt➔R328 0.5 ±1.6***, NtΔ9AA 24.3 ±10.2n.s., ΔCt➔Y350 28.0 ±14.4n.s., NtΔ9AA/ΔCt➔Y350 17.3 ±6.9n.s., NtΔ9AA/ΔCt➔Y350/ΔC341 15.0 ±8.8n.s., and No-NxS 13.6 ±8.3n.s.. ***means significantly different from WT, one-way ANOVA 8 degrees of freedom followed by Scheffe tests, p<0.001. n.s. means not significantly different from WT, p>0.001. EC50 (nM) and maximum response (expressed as a % of mβ2AR::GFP’s maximum response) for isoprenaline are indicated in the table. N/A indicates not applicable, EC50 and maximum response cannot be calculated if the mutant shows no activity in response to isoprenaline and no dose response curve can be fitted, as it is the case here for ΔCt➔R328.
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pone.0141696.g003: Reducing mouse β2AR primary sequence to that of the odorant receptor M71.(A) mβ2AR::GFP showing homogenous and sharp edges staining of OP6 cells (observed in 10/10 cells), is included in small isolated inclusions and also locates to filopodia (arrow head in magnified picture). (B) On the contrary, ΔCt➔R328 shows accumulation around the nucleus and does not locate to filopodia (9/10 cells, magnified picture). (C) The mutated final version of mβ2AR that looks like an OR, NtΔ9AA/ΔCt➔Y350/ΔC341, shows similar localization in OP6 cells as WT, including in filopodia (arrow head in magnified picture). (D) In the same way No-NxS shows proper trafficking in OP6 cells. (E) Dose response curves obtained by the degree of isoprenaline-induction in FLIPR experiments, are depicted as normalized relative fluorescent units (RFUs) as a function of Log [isoprenaline] (M, molar concentration). The same color code is used as in F. (F) The average number of GFP-labeled filopodia per cell is showed (bars in graph, mean +s.d.) as well as the number of cells with ≤1 GFP-labeled filopodia (blue diamonds). WT labels 31.8 ±16.2 filopodia per cell, ΔCt➔R328 0.5 ±1.6***, NtΔ9AA 24.3 ±10.2n.s., ΔCt➔Y350 28.0 ±14.4n.s., NtΔ9AA/ΔCt➔Y350 17.3 ±6.9n.s., NtΔ9AA/ΔCt➔Y350/ΔC341 15.0 ±8.8n.s., and No-NxS 13.6 ±8.3n.s.. ***means significantly different from WT, one-way ANOVA 8 degrees of freedom followed by Scheffe tests, p<0.001. n.s. means not significantly different from WT, p>0.001. EC50 (nM) and maximum response (expressed as a % of mβ2AR::GFP’s maximum response) for isoprenaline are indicated in the table. N/A indicates not applicable, EC50 and maximum response cannot be calculated if the mutant shows no activity in response to isoprenaline and no dose response curve can be fitted, as it is the case here for ΔCt➔R328.

Mentions: First, we internally deleted the Nt of mβ2AR to be a similar length to M71, with only one N-linked glycosylation site. This 9 residues deletion, inside the Nt of mβ2AR (NtΔ9AA; green circles in 2D topology of the protein in Fig 1B), includes one of the glycosylation consensus sequences NxS. We observed normal trafficking and maximum responses to isoprenaline and the half maximal effective concentration (EC50) for NtΔ9AA comparable to WT (i.e. mβ2AR::GFP, Fig 3F and yellow curve in Fig 3E). This indicates that a shorter N-terminus with only one N-linked glycosylation site does not affect affinity of this receptor to isoprenaline or downstream ligand-induced signal transduction.


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)

Reducing mouse β2AR primary sequence to that of the odorant receptor M71.(A) mβ2AR::GFP showing homogenous and sharp edges staining of OP6 cells (observed in 10/10 cells), is included in small isolated inclusions and also locates to filopodia (arrow head in magnified picture). (B) On the contrary, ΔCt➔R328 shows accumulation around the nucleus and does not locate to filopodia (9/10 cells, magnified picture). (C) The mutated final version of mβ2AR that looks like an OR, NtΔ9AA/ΔCt➔Y350/ΔC341, shows similar localization in OP6 cells as WT, including in filopodia (arrow head in magnified picture). (D) In the same way No-NxS shows proper trafficking in OP6 cells. (E) Dose response curves obtained by the degree of isoprenaline-induction in FLIPR experiments, are depicted as normalized relative fluorescent units (RFUs) as a function of Log [isoprenaline] (M, molar concentration). The same color code is used as in F. (F) The average number of GFP-labeled filopodia per cell is showed (bars in graph, mean +s.d.) as well as the number of cells with ≤1 GFP-labeled filopodia (blue diamonds). WT labels 31.8 ±16.2 filopodia per cell, ΔCt➔R328 0.5 ±1.6***, NtΔ9AA 24.3 ±10.2n.s., ΔCt➔Y350 28.0 ±14.4n.s., NtΔ9AA/ΔCt➔Y350 17.3 ±6.9n.s., NtΔ9AA/ΔCt➔Y350/ΔC341 15.0 ±8.8n.s., and No-NxS 13.6 ±8.3n.s.. ***means significantly different from WT, one-way ANOVA 8 degrees of freedom followed by Scheffe tests, p<0.001. n.s. means not significantly different from WT, p>0.001. EC50 (nM) and maximum response (expressed as a % of mβ2AR::GFP’s maximum response) for isoprenaline are indicated in the table. N/A indicates not applicable, EC50 and maximum response cannot be calculated if the mutant shows no activity in response to isoprenaline and no dose response curve can be fitted, as it is the case here for ΔCt➔R328.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4626089&req=5

pone.0141696.g003: Reducing mouse β2AR primary sequence to that of the odorant receptor M71.(A) mβ2AR::GFP showing homogenous and sharp edges staining of OP6 cells (observed in 10/10 cells), is included in small isolated inclusions and also locates to filopodia (arrow head in magnified picture). (B) On the contrary, ΔCt➔R328 shows accumulation around the nucleus and does not locate to filopodia (9/10 cells, magnified picture). (C) The mutated final version of mβ2AR that looks like an OR, NtΔ9AA/ΔCt➔Y350/ΔC341, shows similar localization in OP6 cells as WT, including in filopodia (arrow head in magnified picture). (D) In the same way No-NxS shows proper trafficking in OP6 cells. (E) Dose response curves obtained by the degree of isoprenaline-induction in FLIPR experiments, are depicted as normalized relative fluorescent units (RFUs) as a function of Log [isoprenaline] (M, molar concentration). The same color code is used as in F. (F) The average number of GFP-labeled filopodia per cell is showed (bars in graph, mean +s.d.) as well as the number of cells with ≤1 GFP-labeled filopodia (blue diamonds). WT labels 31.8 ±16.2 filopodia per cell, ΔCt➔R328 0.5 ±1.6***, NtΔ9AA 24.3 ±10.2n.s., ΔCt➔Y350 28.0 ±14.4n.s., NtΔ9AA/ΔCt➔Y350 17.3 ±6.9n.s., NtΔ9AA/ΔCt➔Y350/ΔC341 15.0 ±8.8n.s., and No-NxS 13.6 ±8.3n.s.. ***means significantly different from WT, one-way ANOVA 8 degrees of freedom followed by Scheffe tests, p<0.001. n.s. means not significantly different from WT, p>0.001. EC50 (nM) and maximum response (expressed as a % of mβ2AR::GFP’s maximum response) for isoprenaline are indicated in the table. N/A indicates not applicable, EC50 and maximum response cannot be calculated if the mutant shows no activity in response to isoprenaline and no dose response curve can be fitted, as it is the case here for ΔCt➔R328.
Mentions: First, we internally deleted the Nt of mβ2AR to be a similar length to M71, with only one N-linked glycosylation site. This 9 residues deletion, inside the Nt of mβ2AR (NtΔ9AA; green circles in 2D topology of the protein in Fig 1B), includes one of the glycosylation consensus sequences NxS. We observed normal trafficking and maximum responses to isoprenaline and the half maximal effective concentration (EC50) for NtΔ9AA comparable to WT (i.e. mβ2AR::GFP, Fig 3F and yellow curve in Fig 3E). This indicates that a shorter N-terminus with only one N-linked glycosylation site does not affect affinity of this receptor to isoprenaline or downstream ligand-induced signal transduction.

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.