Intracellular trafficking and assembly of specific Kir3 channel/G protein complexes.
We have previously demonstrated that Kir3.1 channels and Gbeta1gamma2 subunits initially interact in the endoplasmic reticulum (ER). To elucidate the role that anterograde protein trafficking pathways may play in the formation of these complexes, we used dominant negative (DN) mutants of the small G proteins Sar 1 and various compartment-specific Rabs which impede anterograde protein trafficking at different steps. Sar 1 H79G and Rab 1 S25N mutants efficiently blocked the plasma membrane trafficking of the Kir3.1/Kir3.4 complex however they did not block the Gbeta1gamma2/Kir3.1 interaction as measured using bioluminescence resonance energy transfer (BRET). This interaction was also insensitive to the presence of DN Rabs 2, 6, 8, and 11. These results confirm that Gbetagamma/Kir3 complexes form early during channel biosynthesis and trafficking. Using a combination of BRET, protein complementation assays and co-immunoprecipitation, we demonstrate that Gbeta1-4 can interact with Kir3.1 in the absence of Kir3.4. Gbeta5 does not directly interact with the channel but can still be co-immunoprecipitated as part of a larger complex. The interaction between Gbeta and Kir3.1 was selectively fostered by co-expression with different Ggamma subunits. When Ggamma1 or Ggamma11 was co-expressed with eGFP-Gbeta3 or eGFP-Gbeta4, the interaction with the effector was lost. Kir3.2 was capable of interacting with Gbeta1-3 and not Gbeta4 or Gbeta5. These interactions were again fostered by co-expression with Ggamma and were also insensitive to DN Sar 1 or Rab 1. Taken together, our data show that these "precocious" channel/G protein interactions are specific and may have implications beyond their basic function in signalling events.
Robitaille
Mélanie
M
Département de Biochimie, Université de Montréal, Montréal, Québec Canada.
Ramakrishnan
Nitya
N
Baragli
Alessandra
A
Hébert
Terence E
TE
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005455:Fluorescent Antibody Technique; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D005799:Genes, Dominant; D006801:Humans; D007668:Kidney; D020559:Monomeric GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D020761:rab1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; C484274:KCNJ5 protein, human; C094401:KCNJ6 protein, human; D011993:Recombinant Fusion Proteins; C493083:SAR1A protein, human; D020559:Monomeric GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins