Live cell imaging of Gs and the beta2-adrenergic receptor demonstrates that both alphas and beta1gamma7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the beta2-adrenergic receptor.
To visualize and investigate the regulation of the localization patterns of Gs and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. alphas-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of alphas, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of alphas. Functional fluorescent beta1gamma7 dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to beta1 (YFP-N-beta1) and a carboxyl-terminal YFP fragment to gamma7 (YFP-C-gamma7). When expressed together, YFP-N-beta1 and YFP-C-gamma7 produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing alphas-CFP, YFP-N-beta1/YFP-C-gamma7, and the beta2-adrenergic receptor (beta2AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, alphas-CFP and YFP-N-beta1/YFP-C-gamma7 stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with beta2AR-labeled vesicles. Moreover, internalization of beta2AR-GFP, but not alphas-CFP or YFP-N-beta1/YFP-C-gamma7, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the Gs subunits and beta2AR utilize different internalization mechanisms. Subsequent trafficking of the Gs subunits and beta2AR also differed in that vesicles labeled with the Gs subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, co-localization of alphas and beta1gamma7 on these endosomes may indicate a means of recycling specific alphasbetagamma combinations to the plasma membrane.
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822-2623, USA.
Thomas R
TR
Hynes
Stacy M
SM
Mervine
Evan A
EA
Yost
Jonathan L
JL
Sabo
Catherine H
CH
Berlot
D017398:Alternative Splicing; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D051381:Rats; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D018343:Receptors, Adrenergic, beta-2; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D019205:GTP-Binding Protein alpha Subunits, Gs; D007545:Isoproterenol