Delayed internalization and lack of recycling in a beta2-adrenergic receptor fused to the G protein alpha-subunit.
BACKGROUND
Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the N-terminus of the G protein alpha-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 beta2-adrenergic receptor (beta2AR) and Galphas indicated that the fusion to the alpha-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 Galphas-fused beta2AR.


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 beta2AR returned rapidly to the plasma membrane, beta2AR-Galphas did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis.


CONCLUSIONS
The covalent linkage between beta2AR and Galphas 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 Galpha 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.
Di Certo
Maria Grazia
MG
Istituto di Neurobiologia e Medicina Molecolare, CNR, c/o Fondazione Santa Lucia/EBRI, Via del Fosso di Fiorano 64/65, 00143 Rome, Italy. mariagrazia.dicerto@inmm.cnr.it
Batassa
Enrico M
EM
Casella
Ida
I
Serafino
Annalucia
A
Floridi
Aristide
A
Passananti
Claudio
C
Molinari
Paola
P
Mattei
Elisabetta
E
D058666:Adrenergic beta-2 Receptor Agonists; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D008954:Models, Biological; D010766:Phosphorylation; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
D058666:Adrenergic beta-2 Receptor Agonists; D044385:GTP-Binding Protein alpha Subunits; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins