ADP-ribosylation factor 6 as a target of guanine nucleotide exchange factor GRP1.
The GRP1 protein contains a Sec7 homology domain that catalyzes guanine nucleotide exchange on ADP-ribosylation factors (ARF) 1 and 5 as well as a pleckstrin homology domain that binds phosphatidylinositol(3,4,5)P(3), an intermediate in cell signaling by insulin and other extracellular stimuli (Klarlund, J. K., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that both endogenous GRP1 and ARF6 rapidly co-localize in plasma membrane ruffles in Chinese hamster ovary (CHO-T) cells expressing human insulin receptors and COS-1 cells in response to insulin and epidermal growth factor, respectively. The pleckstrin homology domain of GRP1 appears to be sufficient for regulated membrane localization. Using a novel method to estimate GTP loading of expressed HA epitope-tagged ARF proteins in intact cells, levels of biologically active, GTP-bound ARF6 as well as GTP-bound ARF1 were elevated when these ARF proteins were co-expressed with GRP1 or the related protein cytohesin-1. GTP loading of ARF6 in both control cells and in response to GRP1 or cytohesin-1 was insensitive to brefeldin A, consistent with previous data on endogenous ARF6 exchange activity. The ability of GRP1 to catalyze GTP/GDP exchange on ARF6 was confirmed using recombinant proteins in a cell-free system. Taken together, these results suggest that phosphatidylinositol(3,4,5)P(3) may be generated in cell membrane ruffles where receptor tyrosine kinases are concentrated in response to growth factors, causing recruitment of endogenous GRP1. Further, co-localization of GRP1 with ARF6, combined with its demonstrated ability to activate ARF6, suggests a physiological role for GRP1 in regulating ARF6 functions.
Langille
S E
SE
Program in Molecular Medicine, Worcester, Massachusetts 01605, USA.
Patki
V
V
Klarlund
J K
JK
Buxton
J M
JM
Holik
J J
JJ
Chawla
A
A
Corvera
S
S
Czech
M P
MP
D016475:3T3 Cells; D020823:ADP-Ribosylation Factor 1; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D016466:CHO Cells; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D006224:Cricetinae; D004789:Enzyme Activation; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*
D020823:ADP-Ribosylation Factor 1; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D020126:Brefeldin A; D002352:Carrier Proteins; D015852:Eukaryotic Initiation Factor-2; D019204:GTP-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D019869:Phosphatidylinositol 3-Kinases; D011500:Protein Synthesis Inhibitors; D011506:Proteins; C000715348:ARF6 protein, human; C000715727:Arf6 protein, mouse