©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Different ARF Domains Are Required for the Activation of Cholera Toxin and Phospholipase D (*)

(Received for publication, October 26, 1994)

Gui-Feng Zhang (1)(§) Walter A. Patton (1) Fang-Jen S. Lee (1)(¶) Marek Liyanage (2) Joong-Soo Han (2) Sue Goo Rhee (2) Joel Moss (1) Martha Vaughan (1)

From the  (1)Pulmonary-Critical Care Medicine Branch and the (2)Laboratory of Biochemistry, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

ADP-ribosylation factors (ARFs), initially described as activators of cholera toxin ADP-ribosyltransferase activity, regulate intracellular vesicular membrane trafficking and stimulate a phospholipase D (PLD) isoform. ARF-like (ARL) proteins are structurally related to ARFs but do not activate cholera toxin and have relatively little effect on PLD. A new human ARL gene termed hARL1, which shares 57% amino acid identity with hARF1, was identified using a polymerase chain reaction-based cloning method. To determine whether different structural elements are responsible for the activation of the A subunit of cholera toxin and PLD, chimeric proteins were constructed by switching the amino-terminal 73 amino acids of ARF1 and ARL1. The recombinant rL73/F protein, in which the amino-terminal 73 amino acids of ARL1 replaced those of ARF1, activated the A subunit of cholera toxin, whereas the rF73/L protein, in which the NH(2)-terminal 73 amino acids of ARF1 replaced those of ARL1, was inactive. The two chimeric proteins had quite opposite effects on PLD activity. rF73/L activated PLD as effectively as rARF1, whereas rL73/F protein activated PLD only slightly. It appears that the amino-terminal region of ARF1 is not critical for its action as a GTP-dependent activator of cholera toxin, whereas it is necessary for activation of the putative effector enzyme, PLD.


INTRODUCTION

Cholera toxin, a secretory product of Vibrio cholerae that is responsible (in large part) for the devastating diarrheal syndrome characteristic of cholera, exerts its effect on intestinal cells via the ADP-ribosylation of the alpha subunit of a heterotrimeric guanine nucleotide-binding (G) protein, i.e. G(s), the stimulatory G protein of adenylyl cyclase, that serves as a signal-transducing element from surface receptors to intracellular effectors(1, 2, 3) . The ADP-ribosyltransferase activity of the A subunit of cholera toxin (CTA) (^1)is stimulated by a family of 20-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors or ARFs(4, 5, 6) . Sequence comparisons of the ARF proteins and other GTP-binding proteins revealed that ARF proteins have a similar degree of relatedness to the Ras superfamily and the heterotrimeric G protein alpha subunits(7) . The family of ARFs includes the ARF-like proteins (ARLs), which share 30-60% amino acid identity with ARFs(8, 9, 10, 11) . Recombinant ARLs bind and hydrolyze GTP but do not function as cofactors for CTA activation, suggesting that ARLs are not functional homologues of ARFs(8) . Under physiological conditions, ARFs participate in vesicular transport through the Golgi in mammalian cells (12) and are present on both nonclathrin- (13) and clathrin-coated vesicles (14, 15) that are accumulated in the presence of GTPS.

ARF proteins were recently reported to activate phospholipase D (PLD), an enzyme that cleaves phosphatidylcholine (PC) to produce phosphatidic acid (PA) and choline(16, 17) . PA can serve as an effector in several physiological processes including DNA synthesis, cell proliferation, and secretory responses. PA may also be metabolized by PA phosphohydrolase to diacylglycerol, a well characterized activator of protein kinase C. Brown et al.(16) and Cockcroft et al.(17) purified a cytosolic component from bovine brain that markedly enhanced PLD activity in membranes in a GTP-dependent manner. This cytosolic factor was identified as two small GTP-binding proteins, ARF1 and ARF3. Recent work of Massenburg et al.(18) has shown that all three classes of ARFs can activate PLD. The effects of ARL proteins on PLD have not been reported. To determine whether different structural elements are responsible for the two ARF activities, chimeric proteins were constructed by switching the amino-terminal amino acid sequences of ARF1 and ARL1. We report here the identification of different ARF domains involved in the activation of cholera toxin and phospholipase D.


EXPERIMENTAL PROCEDURES

Materials

Enzymes for molecular biology were purchased from Boehringer Mannheim. Rat brains from Bioproducts Inc. or PelFreez Biologicals were stored at -70 °C until use. ARF-stimulatable PLD was partially purified as described using heparin high performance liquid chromatography(18) . Sources of other materials have been published previously(18, 19) .

Isolation of Human ARL1 cDNA

Human ARL1 was isolated initially by polymerase chain reactions (PCR) from a yeast cDNA library (Clontech) that was contaminated with human cDNA. Five PCRs were used to obtain segments of cDNA and to assemble a composite sequence of the full-length coding region. Primers 1222 (5`-TTGACACCAGACCAACTGGTAATG-3`), 1222B (5`-ACCGGCGCTCAGCTGGAATT-3`), 1218 (5`-GGTGGCGACGACTCCTGGAGCCCG-3`), and 1218A (5`-CGTCAGTATCGGCGGAATTC-3`) are complementary to sequences immediately upstream or downstream of the EcoRI insertion site of vector gt11. Degenerate nucleotides (DVGG-R1 and ARF6.5R) correspond to part of the consensus sequence VWDVGGQD and the specific sequence TYKNVKFN in human ARF 6, respectively. The open reading frame was completed using the 5` RACE (rapid amplification of 5` cDNA ends) procedure. Two different human cDNA libraries (human fetal brain and human fibroblast line; Stratagene) served as templates in the one-site-specific PCR used to capture 3` and 5` ends.

Construction of Plasmids for Chimera F73/L and L73/F

To construct chimeric proteins, two DNA fragments (region 1 from amino acid 1 to 73, and region 2 from 74 to 181; Fig. 1) each from ARF1 and ARL1 were amplified from their cDNAs by PCR. Ligation of region 1 of ARF1 and region 2 of ARL 1 produced the chimeric construct F73/L. Ligation of region 1 of ARL1 and region 2 of ARF1 yielded the chimeric construct L73/F. Chimeric inserts were subcloned into the pT7/Nde expression vector(19) . The nucleotide sequences of the two chimeric constructs were verified using a standard dideoxy sequencing method (Sequenase, U. S. Biochemical Corp.). A PCR-generated alteration was found near the COOH terminus in L73/F, where TCC in ARF1 was changed to CCC, which would result in replacement of Ser-174 with Pro.


Figure 1: Comparison of deduced amino acid sequences of hARF1 and hARL1. The human ARL1 cDNA and the predicted protein sequences may be accessed in GenBank using accession no. L28997. Arrow indicates the position of ligation of the two parts of the chimeric proteins, rF73/L and rL73/F.



Expression and Purification of rARF1, rARL1, rF73/L, and rL73/F Proteins

Plasmids were transformed into BL21 (DE3) Escherichia coli. For large scale production of recombinant proteins, 5 ml of overnight culture were used to inoculate 1 liter of LB broth containing carbenicillin (50 µg/ml), followed by shaking at 37 °C. When A reached 0.6-0.8, protein production was induced with 0.5 mM isopropyl-1-thio-beta-D-galactopyranoside for 3 h, and bacteria were collected by centrifugation and stored at -20 °C. Cell pellets were suspended in 10 ml of phosphate-buffered saline (pH 7.4) containing 0.5 mg/ml lysozyme and disrupted by sonication. Insoluble material was pelleted at 100,000 times g and the supernatant was applied to a column (2.5 times 112 cm) of Ultrogel AcA 54, which was eluted with TENDS buffer(19) . Fractions of greatest purity and activity were pooled and stored at -20 °C.

Assay for ARF Stimulation of CTA

Assays were performed in a 200 µl containing the indicated amount of recombinant protein, 50 mM potassium phosphate (pH 7.5), 10 mM MgCl(2), 20 mM dithiothreitol, 0.3 mg/ml ovalbumin, 20 mM agmatine, and 0.003% SDS or 0.3 mg/ml cardiolipin or 3 mM DMPC plus 0.2% sodium cholate, as indicated, 0.2 mM [^14C-adenine] NAD (4-5 times 10^4 cpm), CTA (0.5 µg), and GTPS or GDPbetaS as indicated. After incubation at 30 °C for 1 h, [^14C-adenine]ADP-ribosylagmatine was separated on AG 1-X2 for radioassay.

Assay for ARF Stimulation of PLD

PC hydrolysis was assayed as described in (16) with slight modifications(18) . Assays, in a volume of 300 µl, contained 50 mM Hepes (pH 7.5), 3 mM EGTA, 80 mM KCl, 2.5 mM MgCl(2), 1.6 mM CaCl(2), 10 mM agmatine, 100 µM NAD, 1 mM dithiothreitol, 3.4 µM DPPC, 4.7 µM PIP(2), 53.6 µM phosphatidylethanolamine, CTA (1 µg), 0.4 µCi of [^3H-choline methyl]DPPC (50 Ci mmol), 0.6 µg of partially purified PLD (10 µl, 60 µg/ml), and the indicated amount of recombinant protein with 100 µM GTPS or 100 µM GDPbetaS. This modified assay allows measurement of CTA activity under identical conditions by changing the radiolabel from [^3H-choline]DPPC to [^14C-adenine]NAD for direct comparison of CTA and PLD activation. (It is recognized, of course, that the actual state of the different ARF-enzyme complexes interacting with their respective substrates may be quite different.) After incubation at 37 °C for 1 h, 1 ml of methanol:chloroform:concentrated HCl (50:50:0.5, v/v/v) was added followed by 350 µl of 5 mM EGTA in 1 M HCl. After vortex mixing and centrifugation, 575 µl of the aqueous layer was removed for scintillation counting.


RESULTS AND DISCUSSION

Comparison of Amino Acid Sequences of Human ARL1 and ARF1 and Construction of the Chimeric Proteins

Isolation of the human ARL1 gene resulted from an attempt to isolate an analogue of human ARF6 from yeast using PCR (see ``Experimental Procedures''). The deduced amino acid sequences of hARL1 and hARF1 are 57% identical (Fig. 1). hARL1 is 78% identical to Drosophila ARL1 (8) and 99% identical to the recently cloned rat ARL1 in amino acid sequence(11) .

To define the domains responsible for CTA and PLD activation by ARF, two chimeric recombinant proteins were constructed by switching the amino-terminal 73 amino acids of ARF1 and ARL1 (Fig. 1). The first 73 amino acids contain a region similar to the effector loop found in Ras (7, 20) plus the two conserved nucleotide-binding sequences GXXXXGK and DXXGQ. The rL73/F chimera contains the NH(2)-terminal 73 amino acids of ARL1 (40% of the ARL1 sequence) linked to the COOH-terminal 108 residues of ARF1 (60% of the ARF1 sequence). The rF73/L chimera encodes the NH(2)-terminal 73 amino acids of ARF1 linked to the COOH-terminal 108 residues of ARL1. The chimeras, expressed as nonmyristoylated proteins, were purified from E. coli for use in biochemical studies. (Although it is not known whether ARL proteins are myristoylated in vivo, it is known that myristoylation is not absolutely required for the activation of CTA (18, 21) or PLD (16, 17, 18) by rARF1.)

GTP-dependent Stimulation of ADP-ribosyltransferase Activity of Cholera Toxin by rARF1, rF73/L, and rL73/F Proteins

In the presence of 100 µM GTPS, a nonhydrolyzable GTP analogue, and detergent (SDS), rL73/F enhanced CTA activity in a concentration-dependent manner (similar to the wild type rARF1), whereas rF73/L was inactive (Fig. 2). Activation of CTA was not observed with rL73/F in the presence of 100 µM GDPbetaS (Fig. 2B) or in the absence of added nucleotide (data not shown). Therefore, the activation of CTA by the chimera rL73/F was totally GTP-dependent; rARF1, however, activated slightly in the presence of GDPbetaS (Fig. 2A). It is possible that a fraction of the rARF1 was isolated in a GTP-bound form. In fact, rARF6 has been isolated in an active form with bound GTP(22) .


Figure 2: Stimulation of ADP-ribosyltransferase activity of cholera toxin by rARF1, rF73/L, and rL73/F. Activation of CTA by rARF1 (A), rL73/F (B), and rF73/L (C) was evaluated in the NAD:agmatine ADP-ribosyltransferase assay (25) in 0.003% SDS with 0.1-4.0 µg of ARF. Activity of CTA without added ARF was 2.8 and 2.9 nmol/h, with 100 µM GTPS (closedsymbols) and 100 µM GDPbetaS (opensymbols), respectively. Data are means of values of duplicate assays; variance was less than 10%. The experiments were repeated at least twice.



Activation of CTA by ARF requires phospholipid and/or detergent(6) . In the presence of SDS, the maximal activity of rL73/F was similar to that of rARF1 (Fig. 3A), although the GTPS concentration required for half-maximal activation of CTA (EC) by rL73/F (200 µM) was nearly 40 times that required with rARF1 (5 µM). In the presence of DMPC and sodium cholate or cardiolipin, however, the maximal activity of rL73/F was much higher than that of rARF1 (Fig. 3, B and C). The EC values of GTPS for rARF1 and rL73/F were about 0.1 and 10 µM, respectively. Under all these conditions, rF73/L chimera was inactive, resembling wild type rARL1 (data not shown).


Figure 3: GTP-dependent stimulation of ADP-ribosyltransferase activity of cholera toxin by rARF1, rF73/L, and rL73/F. Assays contained 1 µg of rARF1 (circle), rL73/F (up triangle), or rF73/L (box), the indicated concentration of GTPS, and 0.003% SDS (A), 3 mM DMPC plus 0.2% sodium cholate (B), or 0.3 mg/ml cardiolipin (C). Data are means of values of duplicate assays; variance was less than 10%. The experiments were repeated at least twice.



As the rL73/F chimera, like wild type ARF1, stimulates cholera toxin ADP-ribosyltransferase activity, whereas rF73/L, like wild type ARL1, cannot, the functional domain of ARF that is responsible for cholera toxin activation is apparently not localized in the amino-terminal region (at least not in the first 73 amino acids). This is consistent with the observation that deletion of the amino-terminal 13 amino acids of ARF1 did not affect the ability of ARF to activate CTA(19) , but differs from the previous view that the amino terminus of ARF is critical for its activity based on the ability of a peptide with that sequence to inhibit ARF activation of CTA(23) . We propose that the carboxyl-terminal 60% of ARF contains a structural element that is crucial for its function as a CTA cofactor.

Activation of PLD by rARF1, rF73/L, and rL73/F Proteins

Although the ARF-stimulated PLD has not been purified to homogeneity and its subcellular localization remains to be defined, two major forms of PLD activity have been separated(18) . Proteins solubilized from rat brain membranes with Triton X-100 were applied to a heparin-Sepharose CL-6B column followed by heparin high performance liquid chromatography for the complete separation of the two forms of PLD. One form was completely dependent on sodium oleate; the other was dramatically activated by all three classes of ARF, both myristoylated and non-myristoylated, in the presence of GTPS and PIP(2). Activation of the partially purified ARF-dependent form of PLD by the chimeric proteins was assessed using DPPC as substrate in vesicles containing a mixture of phosphatidylethanolamine, PIP(2), and DPPC(16) .

Under the PLD assay conditions and in the presence of 100 µM GTPS (but not GDPbetaS), CTA was activated by rL73/F and rARF1, but not by rF73/L (data not shown), which is in agreement with the results shown in Fig. 2. In contrast, rF73/L, which was inactive in the CTA assay, activated PLD in the presence of 100 µM GTPS in a concentration-dependent manner (Fig. 4C). The maximal activity of rF73/L was nearly equal to that of rARF1 (Fig. 4, A and C). In the same experiment, rL73/F (which activated CTA) produced only a small degree of activation (Fig. 4B). The activation of PLD was clearly GTP-dependent (Fig. 4), although there was a fraction of rARF1 activity (as shown also in Fig. 2) that was not, which could be explained by the presence in the recombinant ARF protein of tightly bound GTP(22) .


Figure 4: Activation of PLD by rARF1, rF73/L, and rL73/F. Assays contained 0.6 µg of partially purified PLD (10 µl, 0.06 mg/ml), and the indicated amount (0.1-8.0 µg) of rARF1 (A), rL73/F (B), or rF73/L (C) with 100 µM GTPS (closed symbols) or 100 µM GDPbetaS (open symbols). Basal activity of PLD without added ARF was 2.9 and 2.3 pmol/h, with GTPS and GDPbetaS, respectively. Data are means of values of triplicate assays; variance was less than 10%.



It appears that an ARF domain that is important for PLD activation is located in the amino-terminal portion of the protein (at least the first 73 amino acids), although the specific location of the activating structure or the mechanism of activation is still unclear. The putative Ras effector loop is also located in this region(7, 20) . The slight GTP-dependent activity of rL73/F, which was observed also with rARL1 (data not shown), may be related to the fact that ARL1 and ARF1 are 84% identical in amino acids 37-55 (the region similar to the putative Ras effector loop) and 68% identical in sequences of the first 73 amino acids. The finding that rL73/F is clearly less potent than rARF1 may mean that it does not contain all of the specific sequence necessary for optimal PLD activation (at least not for this form of partially purified PLD), or that it associates less well with membranes (phospholipids).

In any case, it seems that different functional domains are involved in the activation of CTA and PLD; thus, ARF could be a rather versatile protein, perhaps subject to control related to subcellular location and/or the presence of effector molecules. The role of PLD activation by ARF in vesicle-mediated membrane trafficking is at present speculative. Activation of PLD by ARF proteins could generate second messengers that are involved in signaling processes or products, e.g. phosphatidic acid, that influence membrane properties and thereby vesicle budding or fusion. The action of PLD, by altering membrane phospholipid composition, might directly or indirectly provide binding sites for membrane docking proteins(16) . Liscovitch et al.(24) have recently published an appealing hypothesis involving a positive feedback between PLD activation and PIP(2) biosynthesis in ARF-regulated vesicle fusion. It is tempting to speculate that different functions of ARF are involved in this action and in its operation as a critical compound required for coatomer or AP-1 binding in vesicle formation. As the physiological role of ARF-stimulated PC hydrolysis and the mechanism of ARF-effector interaction are still unknown, elucidation of structure-function relationships in ARFs and ARLs may lead to a better understanding of the precise roles of ARF and ARL proteins in signaling and other cellular processes.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L28997[GenBank].

§
To whom correspondence should be addressed: Rm. 5N307, Bldg. 10, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-5193; Fax: 301-402-1610; zhangg{at}fido.nhlbi.nih.gov.

Present address: Institute of Molecular Medicine, School of Medicine, National Taiwan University, Taipei, Taiwan, R. O. C.

(^1)
The abbreviations used are: CTA, cholera toxin A subunit; ARF, ADP-ribosylation factor; PLD, phospholipase D; ARL, ARF-like; GTPS, guanosine 5`-3-O-(thio)triphosphate; GDPbetaS, guanosine 5`-2-O-(thio)diphosphate; PC, phosphatidylcholine; DPPC, dipalmitoyl PC; PIP(2), phosphatidylinositol 4,5-biphosphate; PA, phosphatidic acid; rARF1, recombinant ARF1; rARL1, recombinant ARL1; rF73/L, chimeric protein in which the amino-terminal 73 amino acids of ARF1 replaced those of ARL1; rL73/F, chimeric protein in which the amino-terminal 73 amino acids of ARL1 replaced those of ARF1; DMPC, dimyristoyl phosphatidylcholine; PCR, polymerase chain reaction.


REFERENCES

  1. Field, M. (1979) Am. J. Clin. Nutr. 32, 189-196 [Abstract]
  2. Kelly, M. T. (1986) Pediatr. Infect. Dis. 5, S101-S105
  3. Moss, J., and Vaughan, M. (1988) Adv. Enzymol. Relat. Areas Mol. Biol. 61, 303-379 [Medline] [Order article via Infotrieve]
  4. Kahn, R. A., and Gilman, A. G. (1984) J. Biol. Chem. 259, 6228-6234 [Abstract/Free Full Text]
  5. Tsai, S.-C., Noda, M., Adamik, R., Moss, J., and Vaughan, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5139-5142 [Abstract]
  6. Moss, J., and Vaughan, M. (1993) Cell. Signalling 5, 367-379 [Medline] [Order article via Infotrieve]
  7. Price, S. R., Barber, A., and Moss, J. (1990) in ADP-Ribosylating Toxins and G Proteins (Moss, J., and Vaughan, M., eds) pp. 397-424, American Society for Microbiology, Washington, DC
  8. Tamkun, J. W., Kahn, R. A., Kissinger, M., Brizuela, B. J., Rulka, C., Scott, M. P., and Kennison, J. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3120-3124 [Abstract]
  9. Clark, J., Moore, L., Krasinskas, A., Way, J., Battey, J., Tamkun, J., and Kahn, R. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8952-8956 [Abstract]
  10. Cavenagh, M. M., Breiner, M., Schürmann, A., Rosenwald, A. G., Terui, T., Zhang, C.-J., Randazzo, P. A., Adams, M., Joost, H. G., and Kahn, R. A. (1994) J. Biol. Chem. 269, 18937-18942 [Abstract/Free Full Text]
  11. Schürmann, A., Breiner, M., Becker, W., Huppertz, C., Kainulainen, H., Kentrup, H., and Joost, H. G. (1994) J. Biol. Chem. 269, 15683-15688 [Abstract/Free Full Text]
  12. Donaldson, J. G., Kahn, R. A., Lippincott-Schwartz, J., and Klausner, R. D. (1991) Science 254, 1197-1199 [Medline] [Order article via Infotrieve]
  13. Serafini, T., Orci, L., Amherdt, M., Brunner, M., Kahn, R. A., and Rothman, J. E. (1991) Cell 67, 239-253 [Medline] [Order article via Infotrieve]
  14. Lenhard, J. M., Kahn, R. A., and Stahl, P. D. (1992) J. Biol. Chem. 267, 13047-13052 [Abstract/Free Full Text]
  15. Stamnes, M. A., and Rothman, J. E. (1993) Cell 73, 999-1005 [Medline] [Order article via Infotrieve]
  16. Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C., and Sternweis, P. C. (1993) Cell 75, 1137-1144 [Medline] [Order article via Infotrieve]
  17. Cockcroft, S., Thomas, G. M. H., Fensome, A., Geny, B., Cunningham, E., Gout, I., Hiles, I., Totty, N. F., Truong, O., and Hsuan, J. J. (1994) Science 263, 523-526 [Medline] [Order article via Infotrieve]
  18. Massenburg, D., Han, J.-S., Liyanage, M., Patton, W. A., Rhee, S. G., Moss, J., and Vaughan, M. (1994) Proc. Natl. Acad. Sci. U. S. A. , in press
  19. Hong, J.-X., Haun, R. S., Tsai, S.-C., Moss, J., and Vaughan, M. (1994) J. Biol. Chem. 269, 9743-9745 [Abstract/Free Full Text]
  20. Pai, E. F., Kabsch, W., Krengel, U., Holmes, K. C., John, J., and Wittinghofer, A. (1989) Nature 341, 209-214 [CrossRef][Medline] [Order article via Infotrieve]
  21. Haun, R. S., Tsai, S.-C., Adamik, R., Moss, J., and Vaughan, M. (1993) J. Biol. Chem. 268, 7064-7068 [Abstract/Free Full Text]
  22. Welsh, C. F., Moss, J., and Vaughan, M. (1994) J. Biol. Chem. 269, 15583-15587 [Abstract/Free Full Text]
  23. Kahn, R. A., Randazzo, P. A., Serafini, T., Weiss, O., Rulka, C., Clarks, J., Amherdt, M., Roller, P., Orci, L., and Rothman, J. E. (1992) J. Biol. Chem. 267, 13039-13046 [Abstract/Free Full Text]
  24. Liscovitch, M. L., Chalifa, V., Pertile, P., Chen, C.-S., and Cantley, L. C. (1994) J. Biol. Chem. 269, 21403-21406 [Abstract/Free Full Text]
  25. Tsai, S.-C., Noda, M., Adamik, R., Chang, P. P., Chen, H.-C., Moss, J., and Vaughan, M. (1988) J. Biol. Chem. 263, 1768-1772 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.