The N-terminal pleckstrin, coiled-coil, and IQ domains of the exchange factor Ras-GRF act cooperatively to facilitate activation by calcium.
We have recently shown that the neuronal exchange factor p140 Ras-GRF becomes activated in vivo in response to elevated calcium levels [C. L. Farnsworth, N. W. Freshney, L. B. Rosen, A. Ghosh, M. E. Greenberg, and L. A. Feig, Nature (London) 376:524-527, 1995]. Activation is mediated by calcium-induced calmodulin binding to an IQ domain near the N terminus of Ras-GRF. Here we show that the adjacent N-terminal pleckstrin homology (PH), coiled-coil, and IQ domains function cooperatively to allow Ras-GRF activation. Deletion of the N-terminal PH domain redistributes a large percentage of Ras-GRF from the particulate to the cytosolic fraction of cells and renders the protein insensitive to calcium stimulation. A similar cellular distribution and biological activity are observed when only the core catalytic domain is expressed. Although the PH domain is necessary for particulate association of Ras-GRF, it is not sufficient for targeting the core catalytic domain to this cellular location. This requires the PH domain and the adjacent coiled-coil and IQ sequences. Remarkably, this form of Ras-GRF is constitutively activated. The PH and coiled-coil domains must also perform an additional function, since targeting to the particulate fraction of cells is not sufficient to allow Ras-GRF activation by calcium. A Ras-GRF mutant containing the PH domain from Ras-GTPase-activating protein in place of its own N-terminal PH domain localizes to the particulate fraction of cells but does not respond to calcium. Similar phenotypes are seen with mutant Ras-GRFs containing point mutations in either the PH or coiled-coil domain. These findings argue that the N-terminal PH, coiled-coil, and IQ domains of Ras-GRF function together to connect Ras-GRF to multiple components in the particulate fractions of cells that are required for responsiveness of the protein to calcium signaling.
Buchsbaum
R
R
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Telliez
J B
JB
Goonesekera
S
S
Feig
L A
LA
D016475:3T3 Cells; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry*; D002118:Calcium / Q000502:physiology*; D002460:Cell Line; D020662:Guanine Nucleotide Exchange Factors; D051379:Mice; D009474:Neurons; D010750:Phosphoproteins; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D017384:Sequence Deletion; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1
D001798:Blood Proteins; D020662:Guanine Nucleotide Exchange Factors; D010750:Phosphoproteins; D011506:Proteins; C039077:platelet protein P47; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1; D002118:Calcium