A protein binding to a minor-group human rhinovirus (HRV2) was purified from HeLa cell culture supernatant. The amino acid sequences of tryptic peptides showed identity with the human low density lipoprotein (LDL) receptor (LDLR). LDL and HRV2 mutually competed for binding sites on human fibroblasts. Cells down-regulated for LDLR expression yielded much less HRV2 upon infection than cells with up-regulated LDLR. Virus also bound to the large subunit of the a2-macroglobulin receptor/LDLRrelated protein (a2MR/LRP). LDLR-deficient fibroblasts yielded considerably less virus in the presence of receptorassociated protein (RAP), providing evidence that a2MR/LRP also acts as a minor group HRV receptor.Common colds most frequently arise through infection with human rhinoviruses (HRVs). The 102 antigenically distinct serotypes are divided into two groups based on receptor specificity (1, 2). The major group binds to the intercellular adhesion molecule 1 (ICAM-1) (3)(4)(5), and the minor group has been shown to attach to a membrane protein with a relative molecular mass of about 120 kDa (6, 7). ICAM-1 and the poliovirus receptor (8) are members of the immunoglobulin superfamily. As the three-dimensional structures of representative HRVs from the two different receptor groups (9, 10) and of poliovirus (11) show considerable similarity, it might have been expected that the minor group receptor would also belong to this family. However, in this communication we present evidence that minor-group HRVs gain access to the cell via members of the low density lipoprotein (LDL) receptor (LDLR) family (12,13). MATERIALS AND METHODSPurification of HRV2-Binding Protein. Two hundred liters of HeLa cell culture supernatant were concentrated ten times by ultrafiltration, dialyzed against 250 liters of H20 containing 0.02% NaN3, and adjusted to contain 20 mM N-methylpiperazine hydrochloride (pH 4.5). Precipitated material was removed, and the filtered supernatant was applied to a 0.5-liter Macroprep 50 Q column (Bio-Rad). Bound material was eluted with the same buffer containing 0.5 M NaCl. After adjustment to pH 7.2 with 1 M Tris HCl (pH 8), the material was loaded onto a 100-ml Lens culinaris lectin column (Pharmacia), and bound protein was eluted with phosphatebuffered saline (PBS) containing 0.5 M a-D-methyl glucopyranoside and precipitated with (NH4)2SO4 at 50o saturation. The precipitate was dissolved in 200 ml of PBS, the solution was passed over a 40-ml Jacalin agarose column (Vector Laboratories), and bound protein was eluted with 120 ml of 0.1 M a-D-methyl galactopyranoside in PBS and precipitated with (NH4)2SO4 as above. The precipitate was dissolved in 20 mM N-methylpiperazine hydrochloride (pH 4.5) and desalted on a PD-10 column (Pharmacia). Protein was applied onto a Mono Q HR 5/5 column (Pharmacia) and eluted with a gradient of 0-0.5 M NaCl in the same buffer. The binding activity was monitored throughout the purification procedure on ligand blots (7). Active fractions were concentrated to 1.5 ml with a Centricon-30...

The Ras protein signals to a number of distinct pathways by interacting with diverse downstream effectors. Among the effectors of Ras are the Raf kinase and RalGDS, a guanine nucleotide dissociation stimulator specific for Ral. Despite the absence of significant sequence similarities, both effectors bind directly to Ras, but with different specificities. We report here the 2.1 A crystal structure of the complex between Ras and the Ras-interacting domain (RID) of RalGDS. This structure reveals that the beta-sheet of the RID joins the switch I region of Ras to form an extended beta-sheet with a topology similar to that found in the Rap-Raf complex. However, the side chain interactions at the joining junctions of the two interacting systems and the relative orientation of the two binding domains are distinctly different. Furthermore, in the case of the Ras-RID complex a second RID molecule also interacts with a different part of the Ras molecule, the switch II region. These findings account for the cross-talk between the Ras and Ral pathways and the specificity with which Ras distinguishes the two effectors.

The yeast two-hybrid system was used to identif proteins that interact with Ras. The H-Ras protein was found to interact with a g nnucleotide di ostimulator (GDS) that has been previously shown to regulate guanine nudeotide exchange on another member of the Ras protein family, Ral. The interaction is meted by the C-terminal, catalytic segment ofthe RalGDS and can be detected both in vivo, using the two-hybrid system, and in vto, with purfi recombinant proein. The inter of the RaIGDS C-teri segment with Ras is specific, dependent on activation ofRas by GTP, and blocked by a muttion that affects Ras effector fction. These characteristics are similar to those previously demonstrated for the interaction between Ras and its putative effector, Raf, n that the RalGDS may also be a Ras effector. Consistent with this idea, the RaIGDS was found to inhibit the binding of Raf to Ras.The H-ras protooncogene plays a critical role in regulating cell growth, motility, and differentiation, and it is mutationally activated in many types ofcancer (1). The product ofthis gene is a membrane-associated protein that is the prototype of a family of small GTPases. This family includes the Rac and Rho proteins, which regulate cytoskeletal function; the Rab proteins, which are involved in intracellular vesicle transport; the Ran protein, which is involved in nuclear transport and cell cycle control; and the Ral proteins, which are membrane or vesicle-associated proteins of unknown function (2).Each of these proteins functions as a molecular switch, transmitting a signal in the active GTP-bound state and reverting to an inactive state when the bound GTP is hydrolyzed to GDP. The intrinsic GTPase activity ofthese proteins is accelerated by GTPase-activating proteins or GAPs, of which Ras-GAP (p1200AP) is the best characterized (3). The function of the small GTPases is positively regulated by guanine nucleotide exchange factors or dissociation stimulators (GDS proteins), which catalyze the exchange of GDP and GTP (3,4 Two-Hybrid Assays. The yeast reporter strain Y153 (MATa ura3-52 leu2-3,112 his3-200 ade2-101 trpl-901 gal4A gal80A LYS2::GALI-HIS3 GAL)::GALI-lacZ) (17) was used as host. Cells were transformed with Gal4 DNA-binding and activation domain fusion plasmids by using the lithium acetate procedure (18) and plated on selective synthetic medium (0.67% yeast nitrogen base/2% sucrose with appropriate Abbreviations: GAP, GTPase-activating protein; GDS, guanine nucleotide dissociation stimulator; GST, glutathione S-transferase; GMP-PCP, 5'-guanylyl methylenediphosphate.

The RalA and RalB proteins comprise a distinct family of small GTPases [1]. Ral-specific guanine-nucleotide exchange factors such as RalGDS, Rlf and RGL interact with activated Ras and cooperate with Ras in the transformation of murine fibroblasts [2-5]. Thus, the interaction of RalGDS with Ras and the subsequent activation of Ral are thought to constitute a distinct Ras-dependent signaling pathway. The function of Ral is largely unknown. There is circumstantial evidence that Ral may have a function in regulating the cytoskeleton through its interaction with RIP1 (also known as RLIP or RalBP1), a GTPase-activating protein specific for the small GTPases Cdc42 and Rac [6-8]. Ral also binds to phospholipase D (PLD) and thus may play a role in signaling through phospholipids [9]. We have examined endogenous levels of activated, GTP-bound Ral (Ral-GTP) in Rat-2 fibroblasts stimulated with various mitogens. Lysophosphatidic acid (LPA) and epidermal growth factor (EGF), which activate both Ras-dependent and Ras-independent signaling pathways [10,11], rapidly activated Ral. Inhibition of Ras activation by dominant-negative Ras (RasS17N) or pertussis toxin had little effect on Ral-GTP levels, however. Ral was activated by the Ca2+ ionophore ionomycin, and activation by LPA or EGF could be blocked by a phospholipase C (PLC) inhibitor. The results presented here demonstrate a Ca(2+)-dependent mechanism for the activation of Ral.

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