Delayed internalization and lack of recycling in a beta2-adrenergic receptor fused to the G protein alpha-subunit
- Equal contributors
1 Istituto di Neurobiologia e Medicina Molecolare, CNR, c/o Fondazione Santa Lucia/EBRI, Via del Fosso di Fiorano 64/65, 00143 Rome, Italy
2 Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, Università di Roma "La Sapienza", Viale Regina Elena, 324 00161 Rome, Italy
3 EBRI-European Brain Research Institute, Via del Fosso di Fiorano, 64/65, 00143 Rome, Italy
4 Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
5 Istituto di Neurobiologia e Medicina Molecolare, CNR, Tor Vergata, Via del Fosso del Cavaliere, 100 00133 Rome, Italy
6 Dipartimento di Medicina Sperimentale, Via Vetoio, Coppito 2, Università de L'Aquila, 67100 L'Aquila, Italy
7 Laboratory "B", Regina Elena Cancer Institute, Via delle Messi d'Oro, 156 00158 Rome, Italy
8 Istituto di Biologia e Patologia Molecolari, CNR, c/o Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158 Rome, Italy
9 AIRC-Rome Oncogenomic Center (ROC), Via delle Messi d'Oro, 156 00158 Rome, Italy
BMC Cell Biology 2008, 9:56 doi:10.1186/1471-2121-9-56Published: 7 October 2008
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 β2-adrenergic receptor (β2AR) and Gαs 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 Gαs-fused β2AR.
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.
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 β2AR returned rapidly to the plasma membrane, β2AR-Gαs did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis.
The covalent linkage between β2AR and Gαs 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.