Many of the actions of receptor tyrosine kinases are mediated by the protein Ras, including the activation of various downstream serine/threonine kinases and the stimulation of growth and differentiation. The human protein Grb2 binds to ligand-activated growth factor receptors and downstream effector proteins through its Src-homology (SH) domains SH2 and SH3, respectively, and like its homologue from Caenorhabditis elegans, Sem-5, apparently forms part of a highly conserved pathway by which these receptors can control Ras activity. Here we show that the SH3 domains of Grb2 bind to the carboxy-terminal part of hSos1, the human homologue of the Drosophila guanine-nucleotide-releasing factor for Ras, which is essential for control of Ras activity by epidermal growth factor receptor and sevenless. Moreover, a synthetic 10-amino-acid peptide containing the sequence PPVPPR specifically blocks the interaction. These results indicate that the Grb2/hSos1 complex couples activated EGF receptor to Ras signalling.

A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1.

The potent vasodilator action of cyclic GMP-dependent protein kinase (cGK) involves decreasing the Ca

The small G protein ARF1 is involved in the coating of vesicles that bud from the Golgi compartments. Its activation is controlled by as-yet unidentified guanine-nucleotide exchange factors. Gea1, the first ARF exchange factor to be discovered in yeast, is a large protein containing a domain of homology with Sec7, another yeast protein that is also involved in secretion. Here we characterized a smaller human protein (relative molecular mass 47K) named ARNO, which contains a central Sec7 domain that promotes guanine-nucleotide exchange on ARF1. ARNO also contains an amino-terminal coiled-coil motif and a carboxy-terminal pleckstrin-homology (PH) domain. The PH domain mediates an enhancement of ARNO exchange activity by negatively charged phospholipid vesicles supplemented with phosphatidylinositol bisphosphate. The exchange activity of ARNO is not inhibited by brefeldin A, an agent known to block vesicular transport and inhibit the exchange activity on ARF1 in cell extracts. This suggests that a regulatory component which is sensitive to brefeldin A associates with ARNO in vivo, possibly through the amino-terminal coiled-coil. We propose that other proteins with a Sec7 domain regulate different members of the ARF family.