Research article

Delayed internalization and lack of recycling in a beta₂-adrenergic receptor fused to the G protein alpha-subunit

Maria Grazia Di Certo¹ , Enrico M Batassa^2,3 , Ida Casella⁴ , Annalucia Serafino⁵ , Aristide Floridi^6,7 , Claudio Passananti^8,9 , Paola Molinari^{* 4} and Elisabetta Mattei^{* 1,9}

¹Istituto di Neurobiologia e Medicina Molecolare, CNR, c/o Fondazione Santa Lucia/EBRI, Via del Fosso di Fiorano 64/65, 00143 Rome, Italy

²Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, Università di Roma "La Sapienza", Viale Regina Elena, 324 00161 Rome, Italy

³EBRI-European Brain Research Institute, Via del Fosso di Fiorano, 64/65, 00143 Rome, Italy

⁴Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy

⁵Istituto di Neurobiologia e Medicina Molecolare, CNR, Tor Vergata, Via del Fosso del Cavaliere, 100 00133 Rome, Italy

⁶Dipartimento di Medicina Sperimentale, Via Vetoio, Coppito 2, Università de L'Aquila, 67100 L'Aquila, Italy

⁷Laboratory "B", Regina Elena Cancer Institute, Via delle Messi d'Oro, 156 00158 Rome, Italy

⁸Istituto di Biologia e Patologia Molecolari, CNR, c/o Regina Elena Cancer Institute, Via delle Messi d'Oro 156, 00158 Rome, Italy

⁹AIRC-Rome Oncogenomic Center (ROC), Via delle Messi d'Oro, 156 00158 Rome, Italy

author email corresponding author email* Contributed equally

BMC Cell Biology 2008, 9:56doi:10.1186/1471-2121-9-56

Published:	7 October 2008

Abstract

Background

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 Gα_s 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_s-fused β₂AR.

Results

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-Gα_s did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis.

Conclusion

The covalent linkage between β₂AR and Gα_s 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.