Delayed internalization and lack of recycling in a beta<sub>2</sub>-adrenergic receptor fused to the G protein alpha-subunit
<p>Abstract</p> <p>Background</p> <p>Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the rec...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2008-10-01
|
| Series: | BMC Cell Biology |
| Online Access: | http://www.biomedcentral.com/1471-2121/9/56 |
| _version_ | 1811296675622289408 |
|---|---|
| author | Floridi Aristide Serafino Annalucia Casella Ida Batassa Enrico M Di Certo Maria Passananti Claudio Molinari Paola Mattei Elisabetta |
| author_facet | Floridi Aristide Serafino Annalucia Casella Ida Batassa Enrico M Di Certo Maria Passananti Claudio Molinari Paola Mattei Elisabetta |
| author_sort | Floridi Aristide |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β<sub>2</sub>-adrenergic receptor (β<sub>2</sub>AR) and Gα<sub>s </sub>indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gα<sub>s</sub>-fused β<sub>2</sub>AR.</p> <p>Results</p> <p>The rate of agonist-induced internalization, measured as the disappearance of cell surface immunofluorescence in HEK293 cells permanently expressing N-terminus tagged receptors, was reduced three-fold by receptor-G protein fusion. However, both fused and non-fused receptors translocated to the same endocytic compartment, as determined by dual-label confocal analysis of cells co-expressing both proteins and transferrin co-localization.</p> <p>Receptor recycling, determined as the reversion of surface immunofluorescence following the addition of antagonist to cells that were previously exposed to agonist, markedly differed between wild-type and fused receptors. While most of the internalized β<sub>2</sub>AR returned rapidly to the plasma membrane, β<sub>2</sub>AR-Gα<sub>s </sub>did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis.</p> <p>Conclusion</p> <p>The covalent linkage between β<sub>2</sub>AR and Gα<sub>s </sub>does not appear to alter the initial endocytic translocation of the two proteins, although there is reduced efficiency. It does, however, completely disrupt the process of receptor and G protein recycling. We conclude that the physical separation between receptor and Gα is not necessary for the transit to early endosomes, but is an essential requirement for the correct post-endocytic sorting and recycling of the two proteins.</p> |
| first_indexed | 2024-04-13T05:53:07Z |
| format | Article |
| id | doaj.art-4e3a83dd68fe4cc5adbfbc046bd3e930 |
| institution | Directory Open Access Journal |
| issn | 1471-2121 |
| language | English |
| last_indexed | 2024-04-13T05:53:07Z |
| publishDate | 2008-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Cell Biology |
| spelling | doaj.art-4e3a83dd68fe4cc5adbfbc046bd3e9302022-12-22T02:59:42ZengBMCBMC Cell Biology1471-21212008-10-01915610.1186/1471-2121-9-56Delayed internalization and lack of recycling in a beta<sub>2</sub>-adrenergic receptor fused to the G protein alpha-subunitFloridi AristideSerafino AnnaluciaCasella IdaBatassa Enrico MDi Certo MariaPassananti ClaudioMolinari PaolaMattei Elisabetta<p>Abstract</p> <p>Background</p> <p>Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the N-terminus of the G protein α-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the β<sub>2</sub>-adrenergic receptor (β<sub>2</sub>AR) and Gα<sub>s </sub>indicated that the fusion to the α-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Gα<sub>s</sub>-fused β<sub>2</sub>AR.</p> <p>Results</p> <p>The rate of agonist-induced internalization, measured as the disappearance of cell surface immunofluorescence in HEK293 cells permanently expressing N-terminus tagged receptors, was reduced three-fold by receptor-G protein fusion. However, both fused and non-fused receptors translocated to the same endocytic compartment, as determined by dual-label confocal analysis of cells co-expressing both proteins and transferrin co-localization.</p> <p>Receptor recycling, determined as the reversion of surface immunofluorescence following the addition of antagonist to cells that were previously exposed to agonist, markedly differed between wild-type and fused receptors. While most of the internalized β<sub>2</sub>AR returned rapidly to the plasma membrane, β<sub>2</sub>AR-Gα<sub>s </sub>did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis.</p> <p>Conclusion</p> <p>The covalent linkage between β<sub>2</sub>AR and Gα<sub>s </sub>does not appear to alter the initial endocytic translocation of the two proteins, although there is reduced efficiency. It does, however, completely disrupt the process of receptor and G protein recycling. We conclude that the physical separation between receptor and Gα is not necessary for the transit to early endosomes, but is an essential requirement for the correct post-endocytic sorting and recycling of the two proteins.</p>http://www.biomedcentral.com/1471-2121/9/56 |
| spellingShingle | Floridi Aristide Serafino Annalucia Casella Ida Batassa Enrico M Di Certo Maria Passananti Claudio Molinari Paola Mattei Elisabetta Delayed internalization and lack of recycling in a beta<sub>2</sub>-adrenergic receptor fused to the G protein alpha-subunit BMC Cell Biology |
| title | Delayed internalization and lack of recycling in a beta<sub>2</sub>-adrenergic receptor fused to the G protein alpha-subunit |
| title_full | Delayed internalization and lack of recycling in a beta<sub>2</sub>-adrenergic receptor fused to the G protein alpha-subunit |
| title_fullStr | Delayed internalization and lack of recycling in a beta<sub>2</sub>-adrenergic receptor fused to the G protein alpha-subunit |
| title_full_unstemmed | Delayed internalization and lack of recycling in a beta<sub>2</sub>-adrenergic receptor fused to the G protein alpha-subunit |
| title_short | Delayed internalization and lack of recycling in a beta<sub>2</sub>-adrenergic receptor fused to the G protein alpha-subunit |
| title_sort | delayed internalization and lack of recycling in a beta sub 2 sub adrenergic receptor fused to the g protein alpha subunit |
| url | http://www.biomedcentral.com/1471-2121/9/56 |
| work_keys_str_mv | AT floridiaristide delayedinternalizationandlackofrecyclinginabetasub2subadrenergicreceptorfusedtothegproteinalphasubunit AT serafinoannalucia delayedinternalizationandlackofrecyclinginabetasub2subadrenergicreceptorfusedtothegproteinalphasubunit AT casellaida delayedinternalizationandlackofrecyclinginabetasub2subadrenergicreceptorfusedtothegproteinalphasubunit AT batassaenricom delayedinternalizationandlackofrecyclinginabetasub2subadrenergicreceptorfusedtothegproteinalphasubunit AT dicertomaria delayedinternalizationandlackofrecyclinginabetasub2subadrenergicreceptorfusedtothegproteinalphasubunit AT passananticlaudio delayedinternalizationandlackofrecyclinginabetasub2subadrenergicreceptorfusedtothegproteinalphasubunit AT molinaripaola delayedinternalizationandlackofrecyclinginabetasub2subadrenergicreceptorfusedtothegproteinalphasubunit AT matteielisabetta delayedinternalizationandlackofrecyclinginabetasub2subadrenergicreceptorfusedtothegproteinalphasubunit |