The thyrotropin receptor hinge region is not simply a scaffold for the leucine-rich domain but contributes to ligand binding and signal transduction

The glycoprotein hormone receptor hinge region connects the leucine-rich and transmembrane domains. The prevalent concept is that the hinge does not play a significant role in ligand binding and signal transduction. Portions of the hinge are redundant and can be deleted by mutagenesis or are absent in certain species. A minimal hinge will be more amenable to future investigation of its structure and function. We, therefore, combined and progressively extended previous deletions (Delta) in the TSH receptor (TSHR) hinge region (residues 277-418). TSHRDelta287-366, Delta287-371, Delta287-376, and Delta287-384 progressively lost their response to TSH stimulation of cAMP generation in intact cells, consistent with a progressive loss of TSH binding. The longest deletion (TSHRDelta287-384), reducing the hinge region from 141 to 43 amino acids, totally lost both functions. Surprisingly, however, with deletions extending from residues 371-384, constitutive (ligand-independent) activity increased severalfold, reversing the suppressive (inverse agonist) effect of the TSHR extracellular domain. TSHR-activating point mutations I486F and I568T in the first and second extracellular loops (especially the former) had reduced activity on a background of TSHRDelta287-371. In summary, our data support the concept that the TSHR hinge contributes significantly to ligand binding affinity and signal transduction. Residues within the hinge, particularly between positions 371-384, appear involved in ectodomain inverse agonist activity. In addition, the hinge is necessary for functionality of activating mutations in the first and second extracellular loops. Rather than being an inert linker between the leucine-rich and transmembrane domains, the TSHR hinge is a signaling-specificity domain.     

IL-18 receptor beta-induced changes in the presentation of IL-18 binding sites affect ligand binding and signal transduction

IL-18 is a pleiotropic proinflammatory cytokine that is involved in induction of inflammatory mediators, regulation of the cytotoxic activity of NK cells and T cells, and differentiation and activation of both Th1 and Th2 cells. IL-18 signals through its specific cell surface receptor IL-18R, which comprises two subunits: IL-18R alpha and IL-18R beta. IL-18R alpha alone has a weak affinity for IL-18 binding, while the IL-18R alpha/beta complex has a high affinity. By using several anti-IL-18 mAbs and IL-18 binding protein, we have examined whether these site-specific inhibitors could block the binding of IL-18 to IL-18R alpha and to the IL-18R alpha/beta complex. Here we show that IL-18 binding to IL-18R alpha was inhibited by a neutralizing mAb, 125-2H, while binding of IL-18 to the alpha/beta receptor complex was not. This suggests that IL-18R beta-induced conformational changes may occur in IL-18R alpha upon dimerization, leading to changes in the presentation of IL-18 binding sites. Epitope mapping of 125-2H using human-mouse IL-18 chimeras identified a region in IL-18 that was required for 125-2H recognition. This region, as examined by IL-18R binding and functional analysis, appeared to be critical for triggering signal transduction through the heterodimeric receptor.     

The integrity of the conserved ''WS motif'' common to IL-2 and other cytokine receptors is essential for ligand binding and signal transduction.

Recent studies have identified a new family of cytokine receptors, which is primarily characterized by the conservation of periodically interspersed four cysteine residues and the W-S-X-W-S sequence ('WS motif') within the extracellular domain. However, the role of such conserved structures still remains elusive, in particular that of the WS motif. Interleukin-2 (IL-2) is known to play a critical role in the clonal expansion of antigen-stimulated T lymphocytes, and the IL-2 signal is delivered by one of the receptor components, the IL-2 receptor beta (IL-2R beta) chain. The IL-2R beta chain, unlike the IL-2R alpha chain, belongs to this receptor family. In the present study, we analyzed the function of the WS motif of IL-2R beta (Trp194-Ser195-Pro196-Trp197-Ser198) with the use of site-directed mutagenesis. Our results indicate the critical role of the two Trp residues in the proper folding of the IL-2R beta extracellular domain and point to the general functional importance of the WS motif in the new cytokine receptor family.     

Identification of an essential region for growth signal transduction in the cytoplasmic domain of the human interleukin-4 receptor.

Interleukin-4 (IL-4) is a pleiotropic lymphokine which plays an important role in the immune system by regulating proliferation and differentiation of a wide variety of lymphoid and myeloid cells. These biological effects are manifested via binding of IL-4 to specific membrane-associated high affinity receptors. While the IL-4 receptor (IL-4R) cDNA expresses high affinity binding sites when transfected in COS7 cells, its intracellular domain lacks consensus motifs for known signal transducing molecules such as a tyrosine kinase. In this study, we use a DNA deletion approach to explore the mechanism of signal transduction utilized by the human IL-4R cDNA expressed in a murine pro-B cell line, Ba/F3 cells. Using this system, we have identified the critical region of the cytoplasmic domain of human IL-4R for human IL-4-induced transduction of a growth signal in these cells. Our data indicate that the critical region for signal transduction is located between amino acid residues 433-473 numbering from the carboxyl terminus. This region is highly conserved between mouse and human IL-4R but lacks homology with other cytokine receptors. Our studies additionally demonstrate that the cytoplasmic domain is not essential for forming high affinity IL-4-binding sites nor for ligand internalization.     

A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha chain and its function in IL-5-mediated growth signal transduction.

Interleukin-5 (IL-5) regulates the production and function of B cells, eosinophils, and basophils. The IL-5 receptor (IL-5R) consists of two distinct membrane proteins, alpha and beta. The alpha chain (IL-5R alpha) is specific to IL-5. The beta chain is the common beta chain (beta c) of receptors for IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytoplasmic domains of both alpha and beta chains are essential for signal transduction. In this study, we generated cDNAs of IL-5R alpha having various mutations in their cytoplasmic domains and examined the function of these mutants by expressing them in IL-3-dependent FDC-P1 cells. The membrane-proximal proline-rich sequence of the cytoplasmic domain of IL-5R alpha, which is conserved among the alpha chains of IL-5R, IL-3R, and GM-CSF receptor (GM-CSFR), was found to be essential for the IL-5-induced proliferative response, expression of nuclear proto-oncogenes such as c-jun, c-fos, and c-myc, and tyrosine phosphorylation of cellular proteins including JAK2 protein-tyrosine kinase. In addition, analysis using chimeric receptors which consist of the extracellular domain of IL-5R alpha and the cytoplasmic domain of beta c suggested that dimerization of the cytoplasmic domain of beta c may be an important step in activating the IL-5R complex and transducing intracellular growth signals.     

Identification of an essential amino acid motif within the C terminus of the pituitary adenylate cyclase-activating polypeptide type I receptor that is critical for signal transduction but not for receptor internalization

The pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 (PAC1) receptor is a G protein-coupled receptor and class II receptor member. The receptor domains critical for signaling are unknown. To explore the role of the C terminus, truncations of 63 residues (Tr406), 53 residues (Tr416), 49 residues (Tr420), 44 residues (Tr424), and 37 residues (Tr433) were constructed and expressed in NIH/3T3 cells, and immunofluorescence, radioligand binding, adenylyl cyclase (AC) and phospholipase C (PLC) assays were performed. (125)I-PACAP-27 binding (K(d) = 0.6-1.5 nm) for the Tr406 and Tr433 were similar to wild type Hop and Null splice variants (K(d) = approximately 1.1 nm). Although internalization of ligand for both the Tr406 and Tr433 mutants was reduced to 50-60% at 60 min compared with 76-87% for WT, loss of G protein coupling did not account for differences in internalization. Despite similar binding properties Tr406 and Tr416 mutants showed no AC or PLC response. Addition of 14 amino acids distal to HopTr406 resulted in normal AC and PLC responses. Site-directed mutagenesis indicated that Arg(416) and Ser(417) are essential for G protein activation. The proximal C terminus mediates signal transduction, and the distal is involved with internalization. Two residues within the C terminus, Arg(416) and Ser(417) conserved among class II receptors are the likely sites for G protein coupling.     

A distal arginine in oxygen-sensing heme-PAS domains is essential to ligand binding,signal transduction,and structure

To evaluate the contributions of the G(beta)-2 arginine to signal transduction in oxygen-sensing heme-PAS domains, we replaced this residue with alanine in Bradyrhizobium japonicum FixL and examined the results on heme-domain structure, ligand binding, and kinase regulation. In the isolated R220A BjFixL heme-PAS domain, the iron-histidine bond was increased in length by 0.31 A, the heme flattened even without a ligand, and the interaction of a presumed regulatory loop (the FG loop) with the helix of heme attachment was weakened. Binding of carbon monoxide was similar for ferrous BjFixL and R220A BjFixL. In contrast, the level of binding of oxygen was dramatically lower (K(d) approximately 1.5 mM) for R220A BjFixL, and this was manifested as 60- and 3-fold lower on- and off-rate constants, respectively. Binding of cyanide followed the same pattern as binding of oxygen. The catalytic activity was 3-4-fold higher in the "on-state" unliganded forms of R220A BjFixL than in the corresponding BjFixL species. Cyanide regulation of this activity was strongly impaired, but some inhibition was nevertheless preserved. Carbon monoxide and nitric oxide regulation, although weak in BjFixL, were abolished from R220A BjFixL. We conclude that the G(beta)-2 arginine assists in the binding of oxygen to BjFixL but does not accomplish this by stabilizing the oxy form. This arginine is not absolutely required for regulation, although it is important for shifting a pre-existing kinase equilibrium toward the inactive state on binding of regulatory ligands. These findings support a regulatory model in which the heme-PAS domain operates as an ensemble that couples to the kinase rather than a mechanism driven by a single central switch.     

Mannose 6-Phosphate/Insulin-like growth factor II receptor mediates internalization and degradation of leukemia inhibitory factor but not signal transduction.

Leukemia inhibitory factor (LIF) is a multifunctional cytokine belonging to the interleukin-6 subfamily of helical cytokines, all of which use the glycoprotein (gp) 130 subunit for signal transduction. The specific receptor for LIF, gp190, binds this cytokine with low affinity and is also required for signal transduction. We have recently reported that glycosylated LIF produced by transfected Chinese hamster ovary cells also binds to a lectin-like receptor, mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGFII-R) (Blanchard, F., Raher, S., Duplomb, L., Vusio, P., Pitard, V., Taupin, J. L., Moreau, J. F., Hoflack, B., Minvielle, S., Jacques, Y., and Godard, A. (1998) J. Biol. Chem. 273, 20886-20893). The present study shows that (i) mannose 6-phosphate-containing LIF is naturally produced by a number of normal and tumor cell lines; (ii) other cytokines in the interleukin-6 family do not bind to Man-6-P/IGFII-R; and (iii) another unrelated cytokine, macrophage-colony-stimulating factor, is also able to bind to Man-6-P/IGFII-R in a mannose 6-phosphate-sensitive manner. No functional effects or signal transductions mediated by this lectin-like receptor were observed in various biological assays after LIF binding, and mannose 6-phosphate-containing LIF was as active as non-glycosylated LIF. However, mannose 6-phosphate-sensitive LIF binding resulted in rapid internalization and degradation of the cytokine on numerous cell lines, which suggests that Man-6-P/IGFII-R plays an important role in regulating the amounts of LIF available in vivo.     

Mutations in the WSAWSE and cytosolic domains of the erythropoietin receptor affect signal transduction and ligand binding and internalization.

The terminal development of erythroid progenitor cells is promoted in part through the interaction of erythropoietin (EPO) with its cell surface receptor. This receptor and a growing family of related cytokine receptors share homologous extracellular features, including a well-conserved WSXWS motif. To explore the functional significance of this motif in the murine EPO receptor, five WSAWSE mutants were prepared and their signal-transducing, ligand binding, and endocytotic properties were compared. EPO receptors mutated at tryptophan residues (W-232, W-235----G; W-235----G; W-235----F) failed to mediate EPO-induced growth or pp100 phosphorylation, while S-236----T and E-237----K mutants exhibited partial to full activity (50 to 100% of wild-type growth and induced phosphorylation). Ligand affinity was reduced for mutant receptors (two- to fivefold), yet expression at the cell surface for all receptors was nearly equivalent. Also, the ability of mutated receptors to internalize ligand was either markedly reduced or abolished (W-235----F), indicating a role for the WSAWSE region in hormone internalization. Interestingly, receptor forms lacking 97% of the cytosolic domain (no signal-transducing capacity; binding affinity reduced two- to threefold) internalized EPO efficiently. This and all WSAWSE receptor forms studied also mediated specific cross-linking of 125I-EPO to three accessory membrane proteins (M(r)s, 120,000, 105,000, and 93,000). These findings suggest that the WSAWSE domain of the EPO receptor is important for EPO-induced signal transduction and ligand internalization. In contrast, although the cytosolic domain is required for growth signaling, it appears nonessential for efficient endocytosis.     

The AP2 binding site of synaptotagmin 1 is not an internalization signal but a regulator of endocytosis

One characteristic linking members of the synaptotagmin family to endocytosis is their ability to bind the heterotetrameric AP2 complex via their C2B domain. By using CD4/synaptotagmin 1 chimeras, we found that the internalization signal of synaptotagmin 1 lies at the extreme COOH-terminus of the protein and can function in the absence of the C2B domain that contains the AP2 binding site. However, although not essential for internalization, the C2B domain of synaptotagmin 1 appeared to control the recognition of the internalization motif. By mutagenesis, two sites have been identified that modify regulation by the C2B domain in the neuroendocrine PC12 cell line. Mutation of a dilysine motif in the beta sandwich core of the domain eliminates endocytosis. This site is known to be a site of protein-protein interaction. Mutations in the calcium binding region, or in its close proximity, also affect internalization in PC12 cells. In fibroblasts, the C2B domain inhibits the COOH-terminal internalization signal, resulting in an absence of internalization in those cells. Thus, internalization of synaptotagmin 1 is controlled by the presence of a latent internalization signal in the COOH-terminal region and a regulatory region in the C2B domain. We propose that internalization of synaptotagmin 1 is regulated in this way to allow it to couple the processes of endocytosis and calcium-mediated exocytosis in cells of the neuroendocrine lineage.     

Two basic residues of the h-VPAC1 receptor second transmembrane helix are essential for ligand binding and signal transduction

We mutated the vasoactive intestinal peptide (VIP) Asp(3) residue and two VPAC(1) receptor second transmembrane helix basic residues (Arg(188) and Lys(195)). VIP had a lower affinity for R188Q, R188L, K195Q, and K195I VPAC(1) receptors than for VPAC(1) receptors. [Asn(3)] VIP and [Gln(3)] VIP had lower affinities than VIP for VPAC(1) receptors but higher affinities for the mutant receptors; the two basic amino acids facilitated the introduction of the negatively charged aspartate inside the transmembrane domain. The resulting interaction was necessary for receptor activation. 1/[Asn(3)] VIP and [Gln(3)] VIP were partial agonists at VPAC(1) receptors; 2/VIP did not fully activate the K195Q, K195I, R188Q, and R188L VPAC(1) receptors; a VIP analogue ([Arg(16)] VIP) was more efficient than VIP at the four mutated receptors; and [Asn(3)] VIP and [Gln(3)] VIP were more efficient than VIP at the R188Q and R188L VPAC(1) receptors; 3/the [Asp(3)] negative charge did not contribute to the recognition of the VIP(1) antagonist, [AcHis(1),D-Phe(2),Lys(15),Arg(16),Leu(27)] VIP ()/growth hormone releasing factor (8-27). This is the first demonstration that, to activate the VPAC(1) receptor, the Asp(3) side chain of VIP must penetrate within the transmembrane domain, in close proximity to two highly conserved basic amino acids from transmembrane 2.     

Bacterial internalization mediated by beta 1 chain integrins is determined by ligand affinity and receptor density.

Binding of bacteria to beta 1 chain integrin receptors results in either bacterial adherence or uptake by cultured cells (Isberg, 1991). In this report we show that Staphylococcus aureus coated with high affinity ligands for the beta 1 chain integrin family can be internalized efficiently, whereas bacteria coated with low affinity ligands are poorly internalized. Overproduction of the alpha 5 beta 1 integrin increased the efficiency of bacterial internalization, indicating that the uptake efficiency is directly related to the level of expression of the receptor. By using latex beads or S. aureus coated with mAbs directed against the alpha 5 beta 1 integrin, a roughly semi-logarithmic correlation was observed between the affinity of the receptor-ligand interaction and the rate of bacterial internalization. Evidence is presented that high affinity binding of the bacterium allows the microorganism to compete efficiently with receptor-ligand interactions at the basolateral surface of the cell.     

High affinity ligand binding is not essential for granulocyte-macrophage colony-stimulating factor receptor activation.

The high affinity receptor of the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is a heterodimer composed of two members of the cytokine receptor superfamily. GM-CSF binds to the alpha-subunit (GM-R alpha) with low affinity and to the receptor alpha beta complex (GM-R alpha beta) with high affinity. The GM-CSF.GM-R alpha beta complex is responsible for biological activity. Interactions of the N-terminal helix of mouse GM-CSF with mGM-R alpha beta were examined by introducing single alanine substitutions of hydrophilic residues in this region of mGM-CSF. The consequences of these substitutions were evaluated by receptor binding and biological assays. Although all mutant proteins exhibited near wild-type biological activity, most were defective in high affinity receptor binding. In particular, substitution of Glu-21 with alanine abrogated high affinity binding leaving low affinity binding unaffected. Despite near wild-type biological activity, no detectable binding interaction of this mutant with mGM-R beta in the context of mGM-R alpha beta was observed. Cross-linking studies showed an apparent interaction of this mutant protein with mGM-R alpha beta. The deficient receptor binding characteristics and near wild-type biological activity of this mutant protein demonstrate that mGM-CSF receptor activation can occur independently of high affinity binding, suggesting that conformational changes in the receptor induced by mGM-CSF binding generate an active ligand-receptor complex.     

The cytoplasmic domain of the Plasmodium falciparum ligand EBA-175 is essential for invasion but not protein trafficking

The invasion of host cells by the malaria parasite Plasmodium falciparum requires specific protein-protein interactions between parasite and host receptors and an intracellular translocation machinery to power the process. The transmembrane erythrocyte binding protein-175 (EBA-175) and thrombospondin-related anonymous protein (TRAP) play central roles in this process. EBA-175 binds to glycophorin A on human erythrocytes during the invasion process, linking the parasite to the surface of the host cell. In this report, we show that the cytoplasmic domain of EBA-175 encodes crucial information for its role in merozoite invasion, and that trafficking of this protein is independent of this domain. Further, we show that the cytoplasmic domain of TRAP, a protein that is not expressed in merozoites but is essential for invasion of liver cells by the sporozoite stage, can substitute for the cytoplasmic domain of EBA-175. These results show that the parasite uses the same components of its cellular machinery for invasion regardless of the host cell type and invasive form.     

Interaction of neurochondrin with the melanin-concentrating hormone receptor 1 interferes with G protein-coupled signal transduction but not agonist-mediated internalization

Screening of a human brain cDNA library using the C-terminal tail of the melanin-concentrating hormone receptor 1 (MCHR1) as bait in a yeast two-hybrid assay resulted in the identification of the neurite-outgrowth related factor, neurochondrin. This interaction was verified in overlay, pulldown, and co-immunoprecipitation assays. Deletion mapping confined the binding to the C terminus of neurochondrin and to the proximal C terminus of MCHR1, a region known to be involved in G protein binding and signal transduction. This region of the MCHR1 is also able to interact with the actin- and intermediate filament-binding protein, periplakin. Interactions of MCHR1 with neurochondrin and periplakin were competitive, indicating that these two proteins bind to overlapping regions of MCHR1. Although neurochondrin did not interfere with melanin-concentrating hormone-mediated internalization of the receptor, it did inhibit G protein-coupled signal transduction via both Galpha(i/o) and Galpha(q/11) family G proteins as measured by each of melanin-concentrating hormone-induced G protein-activated inwardly rectifying K(+) channel activity of voltage-clamped amphibian oocytes, by calcium mobilization in transfected mammalian cells, and by reduction in the capacity of melanin-concentrating hormone to promote binding of [(35)S]guanosine 5'-3-O-(thio)triphosphate to both Galpha(o1) and Galpha(11). Immunohistochemistry revealed co-expression of neurochondrin and MCHR1 within the rodent brain, suggesting that neurochondrin may be involved in the regulation of MCHR1 signaling and play a role in modulating melanin-concentrating hormone-mediated functions in vivo.     

The ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.

To examine the role of the ligand binding domain of epidermal growth factor receptor in its dimerization, we studied the dimerization of a truncated form of the receptor that resembles v-erbB in that it lacks a ligand binding domain. Receptor dimerization was determined by sedimentation analysis on sucrose density gradients at different concentrations of Triton X-100. At high concentrations of Triton X-100 (0.2%), the truncated receptor occurred as a monomer and displayed low basal autophosphorylation. By contrast, at low concentrations of Triton X-100 (0.01%), it existed as a dimer and exhibited high basal autophosphorylation. The ability of the truncated receptor to dimerize indicates that the ligand binding domain of the epidermal growth factor receptor is not required for receptor dimerization.     

Nucleocytoplasmic shuttling of p53 is essential for MDM2-mediated cytoplasmic degradation but not ubiquitination

As a shuttling protein, p53 is constantly transported through the nuclear pore complex. p53 nucleocytoplasmic transport is carried out by a bipartite nuclear localization signal (NLS) located at its C-terminal domain and two nuclear export signals (NES) located in its N- and C-terminal regions, respectively. The role of nucleocytoplasmic shuttling in p53 ubiquitination and degradation has been a subject of debate. Here we show that the two basic amino acid groups in the p53 bipartite NLS function collaboratively to import p53. Mutations disrupting individual amino acids in the NLS, although causing accumulation of p53 in the cytoplasm to various degrees, reduce but do not eliminate the NLS activity, and these mutants remain sensitive to MDM2 degradation. However, disrupting both parts of the bipartite NLS completely blocks p53 from entering the nucleus and causes p53 to become resistant to MDM2-mediated degradation. Similarly, mutations disrupting four conserved hydrophobic amino acids in the p53 C-terminal NES block p53 export and prohibit it from MDM2 degradation. We also show that colocalization of a nonshuttling p53 with MDM2 either in the nucleus or in the cytoplasm is sufficient for MDM2-induced p53 polyubiquitination but not degradation. Our data provide new insight into the mechanism and regulation of p53 nucleocytoplasmic shuttling and degradation.     

Interleukin-1 (IL-1) receptor antagonist binds to the 80-kDa IL-1 receptor but does not initiate IL-1 signal transduction.

The interleukin-1 receptor antagonist (IL-1ra) is a protein capable of inhibiting receptor binding and biological activities of IL-1 without inducing an IL-1-like response. Equilibrium binding and kinetic experiments show that IL-1ra binds to the 80-kDa IL-1 receptor on the murine thymoma cell line EL4 with an affinity (KD = 150 pM) approximately equal to that of IL-1 alpha and IL-1 beta for this receptor. However, IL-1ra is unable to induce two early events associated with IL-1 activity. Surface-bound IL-1ra does not undergo receptor-mediated internalization, and IL-1ra does not activate the protein kinase activity responsible for down-modulation of the EGF receptor on the murine 3T3 fibroblast cell line. The failure to induce general, early responses characteristic of IL-1 indicates that IL-1ra is unlikely to act as an agonist on any cell expressing the 80-kDa receptor.     

Critical cytoplasmic domains of the common beta subunit of the human GM-CSF, IL-3 and IL-5 receptors for growth signal transduction and tyrosine phosphorylation.

The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3) and interleukin 5 (IL-5) are composed of two distinct subunits, alpha and beta c. The alpha subunits are specific for each cytokine, whereas the beta subunit (beta c) is shared by the three receptors and is an essential component of signal transduction. We have made a series of mutant beta c cDNAs that delete various regions of the cytoplasmic domain and examined the function of these mutants by coexpressing them with the alpha subunit of the human GM-CSF receptor (hGMR) in an IL-3-dependent mouse pro-B cell line BaF3. Two domains in the membrane-proximal portion of beta c were found to be important for transducing the hGM-CSF-mediated growth signals: one domain between Arg456 and Phe487 appears to be essential for proliferation, and the second domain between Val518 and Asp544 enhances the response to GM-CSF, but is not absolutely required for proliferation. The region between Val518 and Leu626 was responsible for major tyrosine phosphorylation of 95 and 60 kDa proteins. Thus, beta c-mediated major tyrosine phosphorylation of these proteins was apparently separated from proliferation. However, the beta 517 mutant lacking residues downstream of Val518 transmitted a herbimycin-sensitive proliferation signal, suggesting that beta 517 still activates a tyrosine kinase(s). We also evaluated the role of the cytoplasmic domain of the GMR alpha subunit and the results suggest that it is involved in the hGM-CSF-mediated signal transduction, but is not essential.(ABSTRACT TRUNCATED AT 250 WORDS)