Differential expression and effects of gp130 cytokines and receptors in prostate cancer cells.

High levels of circulating interleukin-6 (IL6), and possibly neuroendocrine (NE) differentiation, correlate with advanced prostate cancer (PCa). IL6 has many overlapping biological effects with the related gp130 cytokines LIF and OSM that can be explained by the shared usage of the signalling receptor, gp130. We set out to determine whether LIF and OSM can substitute for IL6 in PCa, particularly in relation to neuroendocrine differentiation. Expression analysis of the gp130 cytokines and receptors by RT-PCR, Southern blotting and immunohistochemistry showed that they are widely expressed in LNCaP, DU145 and PC3 cells, but not in normal prostate epithelial PZ-HPV-7 cells. IL6, but not LIF or OSM inhibited proliferation, induced NE differentiation and tyrosine phosphorylation of STAT3 in LNCaP cells. The data suggests that IL6 has a unique role in the progression of PCa.

The single mutation Phe173 --> Ala induces a molten globule-like state in murine interleukin-6.

A series of three aromatic to alanine mutants of recombinant murine interleukin-6 lacking the 22 N-terminal residues (DeltaN22mIL-6) were constructed to investigate the role of these residues in the structure and function of mIL-6. While Y78A and Y97A have activities similar to that of DeltaN22mIL-6, F173A lacks biological activity. F173A retains high levels of secondary structure, as determined by far-UV circular dichroism (CD), but has substantially reduced levels of tertiary structure, as determined by near-UV CD and (1)H NMR spectroscopy. F173A also binds the hydrophobic dye 1-anilino-8-naphthalenesulfonic acid (ANS) over a range of pH values and exhibits noncooperative equilibrium unfolding (as judged by the noncoincidence of monophasic unfolding transitions monitored by far-UV CD and lambda(max), with midpoints of unfolding at 2.6 +/- 0. 1 and 3.5 +/- 0.3 M urea, respectively, and the lack of an observable thermal unfolding transition). These are all properties of molten globule states, suggesting that the loss of activity of F173A results from the disruption of the fine structure of the protein, rather than from the loss of a side chain that is important for ligand-receptor interactions. Surprisingly, under some conditions, this loosened conformation is no more susceptible to proteolytic attack than the parent protein. By analogy with human IL-6, Phe173 in DeltaN22mIL-6 makes multiple interhelical interactions, the removal of which appear to be sufficient to induce a molten globule-like conformation.

Ligand-specific utilization of the extracellular membrane-proximal region of the gp130-related signalling receptors.

The receptor gp130 is used by the interleukin-6 (IL-6)-type cytokines, which include IL-6 and leukaemia-inhibitory factor (LIF). To investigate the role of the three extracellular membrane-proximal fibronectin-type-III-like (FNIII) modules of gp130 and the related receptor for granulocyte colony-stimulating factor (G-CSFR) in cytokine signal transduction we have transfected into murine myeloid M1-UR21 cells the chimaera (GR-FNIII)gp130, which contains the membrane-proximal FNIII modules of the G-CSFR on a gp130 backbone, and its complement, the chimaera (gp130-FNIII)GR. Whereas the binding affinities of (125)I-labelled IL-6 to (GR-FNIII)gp130, or of (125)I-Tyr1,3-G-CSF to (gp130-FNIII)GR, were similar to wild-type gp130 and wild-type G-CSFR, respectively, (125)I-LIF failed to bind with high affinity to (GR-FNIII)gp130. In assays measuring differentiation the (gp130-FNIII)GR cells were fully responsive to G-CSF, whereas the (GR-FNIII)gp130 cells responded fully to the agonistic anti-gp130 monoclonal antibody (mAb) B-S12, but not to IL-6 or LIF. Neutralizing mAbs that recognize the membrane-proximal FNIII modules of gp130 or the G-CSFR differentially interfered with signalling by B-S12, LIF and G-CSF. The data suggest that B-S12 and G-CSF induce the correct orientation or conformation for signalling by the wild-type and chimaeric homodimeric receptors, that the membrane-proximal region of gp130 is important for the correct formation of the signalling IL-6-IL-6 receptor-gp130 complex and that this region is also involved in LIF-dependent receptor heterodimerization and signalling.

Interaction of granulocyte colony-stimulating factor (G-CSF) with its receptor. Evidence that Glu19 of G-CSF interacts with Arg288 of the receptor.

Granulocyte colony-stimulating factor (G-CSF) forms a tetrameric complex with its receptor, comprising two G-CSF and two receptor molecules. The structure of the complex is unknown, and it is unclear whether there are one or two binding sites on G-CSF and the receptor. The immunoglobulin-like domain and the cytokine receptor homologous module of the receptor are involved in G-CSF binding, and Arg288 in the cytokine receptor homologous module is particularly important. To identify residues in G-CSF that interact with Arg288, selected charged residues in G-CSF were mutated to Ala. To clarify whether there are two binding sites, a chimeric receptor was created in which the Ig domain was replaced with that of the related receptor gp130. This chimera bound G-CSF but could not transduce a signal, consistent with failure of dimerization and loss of one binding site. The G-CSF mutants had reduced mitogenic activity on cells expressing wild-type receptor. When tested with the chimeric receptor, all G-CSF mutants except one (E46A) showed reduced binding, suggesting that Glu46 is important for interaction with the Ig domain. On cells expressing R288A receptor, all the G-CSF mutants except E19A showed reduced mitogenic activity, indicating that Glu19 of G-CSF interacts with Arg288 of the receptor.

Physicochemical characterization of an antagonistic human interleukin-6 dimer.

A noncovalently bound dimeric form of recombinant human IL-6 interleukin-6 (IL-6D) was shown to be an antagonist for IL-6 activity, in a STAT3 tyrosine phosphorylation assay using HepG2 cells, under conditions where it does not dissociate into monomeric IL-6 (IL-6M). The fluorescence from Trp157, the single tryptophan residue in the primary sequence of IL-6, is altered in IL-6D, where the wavelength maximum is blue-shifted by 3 nm and the emission intensity is reduced by 30%. These data suggest that Trp157 is close to, but not buried by, the dimer interface. Both IL-6D and IL-6M are compact molecules, as determined by sedimentation velocity analysis, and contain essentially identical levels of secondary and tertiary structure, as determined by far- and near-UV CD, respectively. IL-6D and IL-6M show the same susceptibility to limited proteolytic attack, and exhibit identical far-UV CD-monitored urea-denaturation profiles with the midpoint of denaturation occurring at 6.0 +/- 0.1 M urea. However, IL-6D was found to dissociate prior to the complete unfolding of the protein, with a midpoint of dissociation of 3 M urea, suggesting that dissociation and dimerization occur when the protein is in a partially unfolded state. Based on these results, we suggest that IL-6D is a metastable domain-swapped dimer, comprising two monomeric units where identical helices from each protein chain are swapped through the loop regions at the "top" of the protein (i.e., the region of the protein most distal from the N- and C-termini). Such an arrangement would account for the antagonistic activity of IL-6D. In this model, receptor binding site I, which comprises residues in the A/B loop and the C-terminus of the protein, is free to bind the IL-6 receptor. However, site III, which includes Trp157 and residues in the C/D loop and N-terminal end of helix D, and perhaps site II, which comprises residues in the A and C helices, are no longer able to bind the signal transducing component of the IL-6 receptor complex, gp130.

The immunoglobulin-like module of gp130 is required for signaling by interleukin-6, but not by leukemia inhibitory factor.

The transmembrane protein gp130 is a shared component of the receptor complexes for the interleukin-6 (IL-6)-type cytokines, which include IL-6, leukemia inhibitory factor (LIF) and oncostatin M (OSM). In addition to its role in the generation of high affinity receptors, gp130 is required for signal transduction by these cytokines. In the present study we have examined the role of the N-terminal located, extracellular immunoglobulin (Ig)-like module of gp130 in signal transduction by IL-6 and LIF. We have expressed wild-type human gp130 or three mutants in murine myeloid M1-UR21 cells that lack functional endogenous gp130 but express the IL-6 receptor (IL-6R) and the LIF receptor (LIFR). By measuring cellular responses, such as morphological changes upon differentiation, soft agar colony formation, and induction of tyrosine phosphorylation of the signal transducer and activator of transcription, STAT3, we show that signaling by IL-6, but not LIF, is significantly reduced by mutations in the Ig-like module of gp130. However, the binding of 125I-labeled IL-6 or LIF is not affected by these mutations. We also present evidence that the Ig-like module forms part of the epitope of an anti-gp130 monoclonal antibody that neutralizes the bioactivity of IL-6, but not of LIF or OSM. The data suggest that gp130-activation by IL-6 and LIF requires different regions of gp130, that the Ig-like module of gp130 may be required for IL-6-induced gp130 dimerization, and that the stoichiometry of the high affinity IL-6 receptor-complex differs from those of the receptor-complexes for LIF and OSM.

Identification and characterization of recombinant murine interleukin-6 with a C-terminal pentapeptide extension using capillary reversed phase HPLC-MS and edman degradation.

We have identified a preparation of recombinant murine interleukin-6 (mIL-6) that, in addition to the anticipated product, also contained approximately equal amounts of mIL-6 with a C-terminal pentapeptide extension. The extension mutant was generated by readthrough of the stopcodon, and termination at a second in-frame stopcodon 12 base pairs 3' in the expression vector. Aliquots of the preparation were subjected to proteolytic digestion with Asp-N and Lys-C-endopeptidase. The resultant peptides were separated by reversed-phase capillary HPLC, and analysed using a combination of mass spectrometry and N-terminal sequence analysis. These data revealed a C-terminal pentapeptide (Gln-Gly-Ser-Val-Asp) extension, with the authentic stopcodon being translated as glutamine. The extension mutant was isolated by reversed-phase HPLC and shown to have similar mitogenic activity to mIL-6 on murine hybridoma 7TD1 cells.

Interleukin-6: structure-function relationships.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays a central role in host defense due to its wide range of immune and hematopoietic activities and its potent ability to induce the acute phase response. Overexpression of IL-6 has been implicated in the pathology of a number of diseases including multiple myeloma, rheumatoid arthritis, Castleman's disease, psoriasis, and post-menopausal osteoporosis. Hence, selective antagonists of IL-6 action may offer therapeutic benefits. IL-6 is a member of the family of cytokines that includes interleukin-11, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, and ciliary neurotrophic factor. Like the other members of this family, IL-6 induces growth or differentiation via a receptor-system that involves a specific receptor and the use of a shared signaling subunit, gp130. Identification of the regions of IL-6 that are involved in the interactions with the IL-6 receptor, and gp130 is an important first step in the rational manipulation of the effects of this cytokine for therapeutic benefit. In this review, we focus on the sites on IL-6 which interact with its low-affinity specific receptor, the IL-6 receptor, and the high-affinity converter gp130. A tentative model for the IL-6 hexameric receptor ligand complex is presented and discussed with respect to the mechanism of action of the other members of the IL-6 family of cytokines.

Roles of histidine 31 and tryptophan 34 in the structure, self-association, and folding of murine interleukin-6.

Interleukin-6 (IL-6) is a multifunctional cytokine which is involved in a broad spectrum of activities such as immune defense, hematopoiesis, and the acute phase response, as well as in the pathogenesis of multiple myeloma. A series of murine IL-6 (mIL-6) mutants, H31A, W34A, and H31A/W34A, were constructed to investigate the roles of His31 and Trp34 in the structure, conformational stability, time-dependent aggregation, folding, and spectral properties of mIL-6. The characteristic pH-dependent quenching of fluorescence of mIL-6 at low pH was shown to be caused by an interaction between Trp34 and protonated His31 at low pH and not associated with Trp157. Denaturant-induced equilibrium unfolding experiments monitored by fluorescence and far-UV CD showed that the increased quantum yield and blue shift of the wavelength of the emission maximum observed for mIL-6 at moderate denaturant concentrations were also associated with Trp34, rather than Trp157. The tendency to form aggregation-prone unfolding intermediates, as judged by poor fits to a two-state unfolding mechanism, low m values (slopes of the unfolding curve in the transition region), and the range of denaturant concentrations over which these intermediates formed, was shown to be higher for H31A than mIL-6 but significantly lower for W34A and H31A/W34A. These differences were most pronounced at pH 7.4 and correlated with the tendencies of the proteins to aggregate at high protein concentrations in the absence of denaturant. As judged by the 1H NMR chemical shifts of the aromatic residues, the global conformations of H31A and W34A were not significantly different from that of mIL-6. Nuclear Overhauser effects (NOE) between the side chains of His31 and Trp34 were consistent with the indole side chain of Trp34 being oriented toward the face of the imidazolium side chain of His31, an arrangement consistent with our estimates of a low interaction energy (0.4-0.6 kcal/mol) between these side chains. A shift in the pKa of the His31 side chain in W34A (+0.3 unit) suggested that, in the absence of Trp34, His31 could interact with other residues. Further mutations in this region should yield forms of mIL-6, even less prone to aggregation, which would be more suitable for NMR studies. Mutation of His31 and Trp34 to alanine did not significantly alter the mitogenic activity of the mutants on mouse hybridoma 7TD1 cells, even though the corresponding region of human IL-6 has been shown to be important for biological activity.

Influence of interleukin-6 (IL-6) dimerization on formation of the high affinity hexameric IL-6.receptor complex.

The high affinity interleukin-6 (IL-6) signaling complex consists of IL-6 and two membrane-associated receptor components: a low affinity but specific IL-6 receptor and the affinity converter/signal transducing protein gp130. Monomeric (IL-6M) and dimeric (IL-6D) forms of Escherichia coli-derived human IL-6 and the extracellular ("soluble") portions of the IL-6 receptor (sIL-6R) and gp130 have been purified in order to investigate the effect of IL-6 dimerization on binding to the receptor complex. Although IL-6D has a higher binding affinity for immobilized sIL-6R, as determined by biosensor analysis employing surface plasmon resonance detection, IL-6M is more potent than IL-6D in a STAT3 phosphorylation assay. The difference in potency is significantly less pronounced when measured in the murine 7TD1 hybridoma growth factor assay and the human hepatoma HepG2 bioassay due to time-dependent dissociation at 37 degrees C of IL-6 dimers into active monomers. The increased binding affinity of IL-6D appears to be due to its ability to cross-link two sIL-6R molecules on the biosensor surface. Studies of the IL-6 ternary complex formation demonstrated that the reduced biological potency of IL-6D resulted from a decreased ability of the IL-6D (sIL-6R)2 complex to couple with the soluble portion of gp130. These data imply that IL-6-induced dimerization of sIL-6R is not the driving force in promoting formation of the hexameric (IL-6 IL-6R gp130)2 complex. A model is presented whereby the trimeric complex of IL-6R, gp130, and IL-6M forms before the functional hexamer. Due to its increased affinity for the IL-6R but its decreased ability to couple with gp130, we suggest that a stable IL-6 dimer may be an efficient IL-6 antagonist.

The interleukin-6 (IL-6) partial antagonist (Q159E,T162P)IL-6 interacts with the IL-6 receptor and gp130 but fails to induce a stable hexameric receptor complex.

The extracellular "soluble" domains of the IL-6 receptor (sIL-6R) and gp130 (sgp130) form a hexameric ternary receptor complex together with IL-6, consisting of two molecules of each component. In this report we have investigated the interactions of the partial IL-6 antagonist (Q159E,T162P)IL-6 ((QT)IL-6), with the sIL-6R and sgp130. The kinetic rate constants of the binding of sIL-6R to immobilized monomeric (QT)IL-6 or IL-6 were obtained using an optical biosensor with analysis of the primary data by linear and nonlinear regression. Both methods of analysis showed that, due to a higher off-rate, sIL-6R has lower apparent affinity for (QT)IL-6 than IL-6. The lower affinity of (QT)IL-6 was further confirmed by equilibrium binding measurements at the sensor surface and in solution. Using the biosensor it was also shown that the (QT)IL-6 complex interacts with sgp130, supporting the notion that the biological activity of (QT)IL-6 is mediated via gp130. However, the IL-6 mutant, when incubated with sIL-6R and sgp130, failed to induce a stable hexameric receptor complex, as shown by narrowbore size exclusion chromatography.

NMR studies of the solution properties of recombinant murine interleukin-6.

The effects of solvent, pH and temperature on the 1H-NMR spectra of recombinant murine interleukin-6 (IL-6) are described. Assignments made from two-dimensional homonuclear spectra are presented for resonances of the fifteen aromatic amino-acid side chains. A time-dependent loss of intensity was observed for all resonances in the spectrum of IL-6, probably as a result of aggregation. This aggregation is markedly temperature-dependent. The pKa values of the four histidine residues in murine IL-6 has been measured; one has a value of 5.5, approx. one pH unit less than the value exhibited by the other three. Analysis of the NOESY spectra has allowed a preliminary characterisation of the nature of interactions among the aromatic side chains within the protein fold. 1H and 15N resonances of residues Thr-4 to Val-21 are assigned from three-dimensional 1H-15N correlated spectroscopy, and evidence is presented for these residues comprising a mobile N-terminal tail with little ordered structure. An N-terminal mutant lacking the first 22 residues of the murine IL-6 sequence and known to possess full biological activity was also examined and shown to have essentially retained the tertiary fold of the native molecule.

Use of a biosensor with surface plasmon resonance detection for the determination of binding constants: measurement of interleukin-6 binding to the soluble interleukin-6 receptor.

The interaction of recombinant human interleukin-6 (IL-6) with the soluble extracellular form of its receptor (sIL-6R) has been characterized by the application of expressions developed for quantitative affinity chromatography to results obtained with a biosensor based on surface plasmon resonance detection. First, the interaction of sIL-6R with IL-6 covalently attached to the biosensor-chip was characterized from the dependence of the surface plasmon resonance response upon the concentration of receptor injected into the biosensor. A binding constant for the interaction between sIL-6R and IL-6 was then determined from the biosensor response observed for mixtures of IL-6 and receptor--a procedure that is shown to provide unequivocal characterization of the competing reaction, irrespective of the model used to describe the biphasic interaction between partitioning receptor and immobilized IL-6. A binding constant of 5 x 10(7) M-1 has been obtained for the interaction of sIL-6R with two equivalent and independent sites on an essentially dimeric IL-6 preparation produced using the pUC vector system, and also for the interaction of sIL-6R with a monomeric IL-6 preparation that was univalent in its interaction with receptor.

Structure-function analysis of human IL-6: identification of two distinct regions that are important for receptor binding.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in host defense. It has been predicted that IL-6 may fold as a 4 alpha-helix bundle structure with up-up-down-down topology. Despite a high degree of sequence similarity (42%) the human and mouse IL-6 polypeptides display distinct species-specific activities. Although human IL-6 (hIL-6) is active in both human and mouse cell assays, mouse IL-6 (mIL-6) is not active on human cells. Previously, we demonstrated that the 5 C-terminal residues of mIL-6 are important for activity, conformation, and stability (Ward LD et al., 1993, Protein Sci 2:1472-1481). To further probe the structure-function relationship of this cytokine, we have constructed several human/mouse IL-6 hybrid molecules. Restriction endonuclease sites were introduced and used to ligate the human and mouse sequences at junction points situated at Leu-62 (Lys-65 in mIL-6) in the putative connecting loop AB between helices A and B, at Arg-113 (Val-117 in mIL-6) at the N-terminal end of helix C, at Lys-150 (Asp-152 in mIL-6) in the connecting loop CD between helices C and D, and at Leu-178 (Thr-180 in mIL-6) in helix D. Hybrid molecules consisting of various combinations of these fragments were constructed, expressed, and purified to homogeneity. The conformational integrity of the IL-6 hybrids was assessed by far-UV CD. Analysis of their biological activity in a human bioassay (using the HepG2 cell line), a mouse bioassay (using the 7TD1 cell line), and receptor binding properties indicates that at least 2 regions of hIL-6, residues 178-184 in helix D and residues 63-113 in the region incorporating part of the putative connecting loop AB through to the beginning of helix C, are critical for efficient binding to the human IL-6 receptor. For human IL-6, it would appear that interactions between residues Ala-180, Leu-181, and Met-184 and residues in the N-terminal region may be critical for maintaining the structure of the molecule; replacement of these residues with the corresponding 3 residues in mouse IL-6 correlated with a significant loss of alpha-helical content and a 200-fold reduction in activity in the mouse bioassay. A homology model of mIL-6 based on the X-ray structure of human granulocyte colony-stimulating factor is presented.

High affinity interleukin-6 receptor is a hexameric complex consisting of two molecules each of interleukin-6, interleukin-6 receptor, and gp-130.

The high affinity human interleukin-6 (IL-6) receptor complex consists of IL-6 and two membrane-associated receptor components, the IL-6 receptor (alpha-subunit) and the high affinity converter and signal transducing molecule, gp-130 (beta-subunit). Recombinant IL-6 and the extracellular ("soluble") components of the IL-6 receptor (sIL-6R) and gp-130 (sgp-130) have been prepared in order to investigate the stoichiometry and binding of these components in the low affinity (IL-6.sIL-6R) and high affinity (IL-6.sIL-6R.sgp-130) IL-6 receptor complexes. Using a combination of size-exclusion chromatography and analytical ultracentrifugation analysis, in the low affinity receptor complex, IL-6 was shown to bind sIL-6R in a stoichiometric ratio of 1:1, whereas the high affinity ternary complex is hexameric consisting of two molecules each of IL-6, sIL-6R, and sgp-130. This is the first direct demonstration of a higher order arrangement for receptor cytokine interactions that exhibit both high and low affinity complexes.

Role of the C-terminus in the activity, conformation, and stability of interleukin-6.

Two murine interleukin-6 (mIL-6) variants were constructed using the polymerase chain reaction (PCR), one lacking the last five residues (183-187) at the C-terminus (pMC5) and another with the last five residues of mIL-6 substituted by the corresponding residues of human IL-6 (pMC5H). The growth stimulatory activity of pMC5 on the mouse hybridoma cell line 7TD1 was < 0.05% of mIL-6, whereas pMC5H and mIL-6 were equipotent. The loss of biological activity of pMC5 correlated with its negligible receptor binding affinity on 7TD1 cells, while the binding of pMC5H was comparable to that of mIL-6. Both pMC5 and pMC5H, like mIL-6, failed to interact with recombinant soluble human IL-6 receptor when assayed by surface plasmon resonance-based biosensor analysis. These studies suggest that the C-terminal seven amino acids of human IL-6, alone, do not define species specificity for receptor binding. A variety of biophysical techniques, as well as the binding of a conformational-specific monoclonal antibody, indicated that the global fold of the mIL-6 variants was similar to that of mIL-6, although small changes in the NMR spectra, particularly for pMC5, were observed. Some of these changes involved residues widely separated in the primary structure. For instance, interactions involving Tyr-22 were influenced by the C-terminal amino acids suggesting that the N- and C-termini of mIL-6 are in close proximity. Equilibrium unfolding experiments indicated that pMC5 was 0.8 kcal/mol less stable than mIL-6, whereas pMC5H was 1.4 kcal/mol more stable. These studies emphasize the structural importance of the C-terminal amino acids of IL-6 and suggest that truncation or mutation of this region could lead to small but significant alterations in other regions of the molecule.

Micropreparative ion exchange high performance liquid chromatography: applications to microsequence analysis.

Data on the characterization of anion and cation micropreparative (50 x 1.6 mm i.d.) HPLC columns is presented. It is shown how subnanomole quantities of protein can be efficiently recovered from such columns, rendering them compatible for use in multidimensional chromatographic strategies for the purification of trace biological samples. By selection of appropriate solvent systems (e.g., buffer-free sodium chloride solutions), the small eluant peak volumes can be loaded directly onto the gas phase sequencer, and N-terminal sequence data obtained. The potential of the technique is illustrated for the purification of a GTPase activating protein (GAP-3).

The purification of a Rap1 GTPase-activating protein from bovine brain cytosol.

Two GTPase-activating proteins (GAPs) have been detected in extracts from bovine brain: GAP-1, which is specific for the activation of ras GTPases, and GAP-3, which is specific for the activation of the rap1 GTPases. We present a strategy for the purification to homogeneity of a cytosolic form of GAP-3 from bovine brain. The 100,000 x g supernatant from homogenized brains was chromatographed sequentially on DEAE Fast Flow, green H-E4BD Sepharose, Bio-Gel A1.5, hydroxyapatite, and phenyl-Sepharose prior to high resolution separation on Mono Q HR 5/5, phenyl-Superose HR 5/5, Mono Q PC 1.6/5, and Superose 12 PC 3.2/30. This procedure resulted in an approximately 18,000-fold purification, yielding 50 micrograms of GAP-3 from 1.6 kg of tissue. Purified cytosolic GAP-3 migrated as a single band of apparent Mr 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, on gel filtration cytosolic GAP-3 chromatographed as a dimer with an apparent Mr 92,000. Purified GAP-3 does not activate ras or rho GTPases and possesses no intrinsic GTPase activity. Amino acid sequence data indicated a proline-rich N terminus. The amino acid sequences of peptides generated by Staphylococcus aureus V8 digestion of reduced and pyridine-ethylated GAP-3 showed no similarity to the predicted primary structure of GAP-1 or any other proteins in the nucleic acid or protein data bases. By comparison with the data of Rubinfeld et al. (Rubinfeld, B., Munemitsu, S., Clark, R., Conroy, L., Watt, K., Crosier, W.J., McCormick, F., and Polakis, P. (1991) Cell 65, 1033-1042), it appears that the membrane-associated (Mr 85,000-95,000) and cytosolic forms of GAP-3 are derived from equivalent, or closely related, genes.