Phosducin-like protein regulates G-protein betagamma folding by interaction with tailless complex polypeptide-1alpha: dephosphorylation or splicing of PhLP turns the switch toward regulation of Gbetagamma folding.
Phosducin-like protein (PhLP) exists in two splice variants PhLP(LONG) (PhLP(L)) and PhLP(SHORT) (PhLP(S)). Whereas PhLP(L) directly inhibits Gbetagamma-stimulated signaling, the G betagamma-inhibitory mechanism of PhLP(S) is not understood. We report here that inhibition of Gbetagamma signaling in intact HEK cells by PhLP(S) was independent of direct Gbetagamma binding; however, PhLP(S) caused down-regulation of Gbeta and Ggamma proteins. The down-regulation was partially suppressed by lactacystine, indicating the involvement of proteasomal degradation. N-terminal fusion of Gbeta or Ggamma with a dye-labeling protein resulted in their stabilization against down-regulation by PhLP(S) but did not lead to a functional rescue. Moreover, in the presence of PhLP(S), stabilized Ggamma subunits did not coprecipitate with stabilized Gbeta subunits, suggesting that PhLP(S) might interfere with Gbetagamma folding. PhLP(S) and several truncated mutants of PhLP(S) interacted with the subunit tailless complex polypeptide-1alpha (TCP-1alpha) of the CCT chaperonin complex, which is involved in protein folding. Knock-down of TCP-1alpha in HEK cells by small interfering RNA also led to down-regulation of Gbetagamma. We therefore conclude that the strong inhibitory action of PhLP(S) on Gbetagamma signaling is the result of a previously unrecognized mechanism of Gbetagamma-regulation, inhibition of Gbetagamma-folding by interference with TCP-1alpha.
Humrich
Jan
J
Institute of Pharmacology and Toxicology, University of Wuerzburg, Germany.
Bermel
Christina
C
Bünemann
Moritz
M
Härmark
Linda
L
Frost
Robert
R
Quitterer
Ursula
U
Lohse
Martin J
MJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047390:Casein Kinase II / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008954:Models, Biological; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D018076:DNA, Complementary; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D046912:Multiprotein Complexes; D009419:Nerve Tissue Proteins; C098789:PDCL protein, human; D020033:Protein Isoforms; D021122:Protein Subunits; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D047390:Casein Kinase II; D056404:Chaperonin Containing TCP-1; D018833:Chaperonins