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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.


A new loss-of-function mutants for activity which retains trafficking.(A) A complete deletion of the Ct of mβ2AR to R328 blocks proper trafficking of the protein, which appears to be retained in the periphery of the nucleus (ΔCt➔R328, 9/10 cells) and does not traffic to filopodia (magnified pictures). (B) The mutation Y326A rescues trafficking of the protein in the cell (Y326A/ΔCt➔R328, 10/10 cells) and to filopodia (arrow head in magnified pictures). (C and D) This rescue is not observed anymore when the 2 N-linked glycosylation sites located in the Nt are invalidated (no NxS / Y326A/ΔCt➔R328) or when the Nt is replaced by the one of M71 (M71 Nt / Y326A/ΔCt➔R328) (respectively 3/10 cells and 8/10 cells). In both cases the protein fails to traffic to filopodia (magnified pictures). (E) Dose response curves to isoprenaline obtained by FLIPR analysis are showed as normalized relative fluorescent units (RFUs) as a function of Log [isoprenaline] (M). 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 filopodium (blue diamonds). ΔCt➔R328 labels only 0.5 ±1.6*** filopodia per cell, whereas Y326A/ΔCt➔R328 26.4 ±15.2 n.s., NtΔ9AA/Y326A/ΔCt➔R328 14.7 ±9.5 n.s., no NxS / Y326A/ΔCt➔R328 4.2 ±4.0***, M71 Nt / Y326A/ΔCt➔R328 0.5 ±1.1*** and Y326A 14.9 ±7.3 n.s.. ***means significantly different from WT, one-way ANOVA 6 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. (G) Snake plot of the 7th and last transmembrane domain and the Ct of the protein showing the localization of the NPxxY region as well as R328.
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pone.0141696.g006: A new loss-of-function mutants for activity which retains trafficking.(A) A complete deletion of the Ct of mβ2AR to R328 blocks proper trafficking of the protein, which appears to be retained in the periphery of the nucleus (ΔCt➔R328, 9/10 cells) and does not traffic to filopodia (magnified pictures). (B) The mutation Y326A rescues trafficking of the protein in the cell (Y326A/ΔCt➔R328, 10/10 cells) and to filopodia (arrow head in magnified pictures). (C and D) This rescue is not observed anymore when the 2 N-linked glycosylation sites located in the Nt are invalidated (no NxS / Y326A/ΔCt➔R328) or when the Nt is replaced by the one of M71 (M71 Nt / Y326A/ΔCt➔R328) (respectively 3/10 cells and 8/10 cells). In both cases the protein fails to traffic to filopodia (magnified pictures). (E) Dose response curves to isoprenaline obtained by FLIPR analysis are showed as normalized relative fluorescent units (RFUs) as a function of Log [isoprenaline] (M). 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 filopodium (blue diamonds). ΔCt➔R328 labels only 0.5 ±1.6*** filopodia per cell, whereas Y326A/ΔCt➔R328 26.4 ±15.2 n.s., NtΔ9AA/Y326A/ΔCt➔R328 14.7 ±9.5 n.s., no NxS / Y326A/ΔCt➔R328 4.2 ±4.0***, M71 Nt / Y326A/ΔCt➔R328 0.5 ±1.1*** and Y326A 14.9 ±7.3 n.s.. ***means significantly different from WT, one-way ANOVA 6 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. (G) Snake plot of the 7th and last transmembrane domain and the Ct of the protein showing the localization of the NPxxY region as well as R328.

Mentions: Our ΔCt truncation mutants show that the mβ2AR Ct tail is involved in G-protein coupling. However, these mutants do not unravel the reason why plasma membrane trafficking fails to occur in ΔCt➔R328, ΔCt➔I334, ΔCt➔A335, and ΔCt➔F336 (Figs 3B, 3F, 6A and 6F). One possible explanation is that the protein needs a specific sequence motif in the Ct tail for proper folding, but the fusion protein still has a cytoplasmic tail from the linker and GFP tag and their sequences are not markedly different from those of mβ2AR. Another possibility is that the mβ2AR protein contains a retention signal sequence that is usually masked by the Ct tail [27]. We therefore looked for a conserved motif near the mβ2AR Ct that might act as a retention sequence. At the end of the last trans-membrane domain of mβ2AR is a highly conserved NPxxY region (NPLIY, Figs 1A and 6G). This region, highly conserved throughout the GPCR superfamily, was shown to be involved in internalization of several GPCRs to the endosomes [20]. Alanine scanning mutagenesis of the three conserved residues reveal that only the isolated substitution of Y326A alters trafficking of the protein to the membrane without the presence of a C-terminus (Neither APLIY, NALIY, nor AALIA can rescue the non-trafficking phenotype). The Y326A/ΔCt➔R328 mutant shows expression similar to mouse WT (Fig 6B and 6F). Interestingly, both ΔCt➔R328 and Y326A/ΔCt➔R328 show no capacity to signal after isoprenaline exposure (Fig 6E, comparable to no plasmid-mock transfection). Thus, we have revealed a major role of the tyrosine residue in the context of the conserved region NPxxY as a retention signal, if exposed, and we describe a new loss-of-function mutant Y326A/ΔCt➔R328, which retains normal plasma membrane trafficking.


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)

A new loss-of-function mutants for activity which retains trafficking.(A) A complete deletion of the Ct of mβ2AR to R328 blocks proper trafficking of the protein, which appears to be retained in the periphery of the nucleus (ΔCt➔R328, 9/10 cells) and does not traffic to filopodia (magnified pictures). (B) The mutation Y326A rescues trafficking of the protein in the cell (Y326A/ΔCt➔R328, 10/10 cells) and to filopodia (arrow head in magnified pictures). (C and D) This rescue is not observed anymore when the 2 N-linked glycosylation sites located in the Nt are invalidated (no NxS / Y326A/ΔCt➔R328) or when the Nt is replaced by the one of M71 (M71 Nt / Y326A/ΔCt➔R328) (respectively 3/10 cells and 8/10 cells). In both cases the protein fails to traffic to filopodia (magnified pictures). (E) Dose response curves to isoprenaline obtained by FLIPR analysis are showed as normalized relative fluorescent units (RFUs) as a function of Log [isoprenaline] (M). 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 filopodium (blue diamonds). ΔCt➔R328 labels only 0.5 ±1.6*** filopodia per cell, whereas Y326A/ΔCt➔R328 26.4 ±15.2 n.s., NtΔ9AA/Y326A/ΔCt➔R328 14.7 ±9.5 n.s., no NxS / Y326A/ΔCt➔R328 4.2 ±4.0***, M71 Nt / Y326A/ΔCt➔R328 0.5 ±1.1*** and Y326A 14.9 ±7.3 n.s.. ***means significantly different from WT, one-way ANOVA 6 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. (G) Snake plot of the 7th and last transmembrane domain and the Ct of the protein showing the localization of the NPxxY region as well as R328.
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Related In: Results  -  Collection

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Show All Figures
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pone.0141696.g006: A new loss-of-function mutants for activity which retains trafficking.(A) A complete deletion of the Ct of mβ2AR to R328 blocks proper trafficking of the protein, which appears to be retained in the periphery of the nucleus (ΔCt➔R328, 9/10 cells) and does not traffic to filopodia (magnified pictures). (B) The mutation Y326A rescues trafficking of the protein in the cell (Y326A/ΔCt➔R328, 10/10 cells) and to filopodia (arrow head in magnified pictures). (C and D) This rescue is not observed anymore when the 2 N-linked glycosylation sites located in the Nt are invalidated (no NxS / Y326A/ΔCt➔R328) or when the Nt is replaced by the one of M71 (M71 Nt / Y326A/ΔCt➔R328) (respectively 3/10 cells and 8/10 cells). In both cases the protein fails to traffic to filopodia (magnified pictures). (E) Dose response curves to isoprenaline obtained by FLIPR analysis are showed as normalized relative fluorescent units (RFUs) as a function of Log [isoprenaline] (M). 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 filopodium (blue diamonds). ΔCt➔R328 labels only 0.5 ±1.6*** filopodia per cell, whereas Y326A/ΔCt➔R328 26.4 ±15.2 n.s., NtΔ9AA/Y326A/ΔCt➔R328 14.7 ±9.5 n.s., no NxS / Y326A/ΔCt➔R328 4.2 ±4.0***, M71 Nt / Y326A/ΔCt➔R328 0.5 ±1.1*** and Y326A 14.9 ±7.3 n.s.. ***means significantly different from WT, one-way ANOVA 6 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. (G) Snake plot of the 7th and last transmembrane domain and the Ct of the protein showing the localization of the NPxxY region as well as R328.
Mentions: Our ΔCt truncation mutants show that the mβ2AR Ct tail is involved in G-protein coupling. However, these mutants do not unravel the reason why plasma membrane trafficking fails to occur in ΔCt➔R328, ΔCt➔I334, ΔCt➔A335, and ΔCt➔F336 (Figs 3B, 3F, 6A and 6F). One possible explanation is that the protein needs a specific sequence motif in the Ct tail for proper folding, but the fusion protein still has a cytoplasmic tail from the linker and GFP tag and their sequences are not markedly different from those of mβ2AR. Another possibility is that the mβ2AR protein contains a retention signal sequence that is usually masked by the Ct tail [27]. We therefore looked for a conserved motif near the mβ2AR Ct that might act as a retention sequence. At the end of the last trans-membrane domain of mβ2AR is a highly conserved NPxxY region (NPLIY, Figs 1A and 6G). This region, highly conserved throughout the GPCR superfamily, was shown to be involved in internalization of several GPCRs to the endosomes [20]. Alanine scanning mutagenesis of the three conserved residues reveal that only the isolated substitution of Y326A alters trafficking of the protein to the membrane without the presence of a C-terminus (Neither APLIY, NALIY, nor AALIA can rescue the non-trafficking phenotype). The Y326A/ΔCt➔R328 mutant shows expression similar to mouse WT (Fig 6B and 6F). Interestingly, both ΔCt➔R328 and Y326A/ΔCt➔R328 show no capacity to signal after isoprenaline exposure (Fig 6E, comparable to no plasmid-mock transfection). Thus, we have revealed a major role of the tyrosine residue in the context of the conserved region NPxxY as a retention signal, if exposed, and we describe a new loss-of-function mutant Y326A/ΔCt➔R328, which retains normal plasma membrane trafficking.

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.