RGS4 and RGS2 bind coatomer and inhibit COPI association with Golgi membranes and intracellular transport.
COPI, a protein complex consisting of coatomer and the small GTPase ARF1, is an integral component of some intracellular transport carriers. The association of COPI with secretory membranes has been implicated in the maintenance of Golgi integrity and the normal functioning of intracellular transport in eukaryotes. The regulator of G protein signaling, RGS4, interacted with the COPI subunit beta'-COP in a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound purified recombinant beta'-COP in vitro. Endogenous cytosolic RGS4 from NG108 cells and RGS2 from HEK293T cells cofractionated with the COPI complex by gel filtration. Binding of beta'-COP to RGS4 occurred through two dilysine motifs in RGS4, similar to those contained in some aminoglycoside antibiotics that are known to bind coatomer. RGS4 inhibited COPI binding to Golgi membranes independently of its GTPase-accelerating activity on G(ialpha). In RGS4-transfected LLC-PK1 cells, the amount of COPI in the Golgi region was considerably reduced compared with that in wild-type cells, but there was no detectable difference in the amount of either Golgi-associated ARF1 or the integral Golgi membrane protein giantin, indicating that Golgi integrity was preserved. In addition, RGS4 expression inhibited trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and impaired secretion of placental alkaline phosphatase from HEK293T cells. The inhibitory effect of RGS4 in these assays was independent of GTPase-accelerating activity but correlated with its ability to bind COPI. Thus, these data support the hypothesis that these RGS proteins sequester coatomer in the cytoplasm and inhibit its recruitment onto Golgi membranes, which may in turn modulate Golgi-plasma membrane or intra-Golgi transport.
Sullivan
B M
BM
Renal Unit, Program in Membrane Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.
Harrison-Lavoie
K J
KJ
Marshansky
V
V
Lin
H Y
HY
Kehrl
J H
JH
Ausiello
D A
DA
Brown
D
D
Druey
K M
KM
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D020755:Coat Protein Complex I / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D020756:Coatomer Protein / Q000378:metabolism*; D016384:Consensus Sequence; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D021122:Protein Subunits; D020710:RGS Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D016415:Sequence Alignment; D013234:Stem Cells / Q000378:metabolism; D014162:Transfection
D020755:Coat Protein Complex I; D020756:Coatomer Protein; D021122:Protein Subunits; D020710:RGS Proteins; D011994:Recombinant Proteins; C105345:Rgs2 protein, mouse; C098799:RGS4 protein; D000469:Alkaline Phosphatase