©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Selective G Protein Coupling by C-C Chemokine Receptors (*)

(Received for publication, September 18, 1995; and in revised form, December 27, 1995)

Yanan Kuang (2) Yanping Wu (1) Huiping Jiang (1) Dianqing Wu (1) (3)(§)

From the  (1)Departments of Pharmacology and Physiology, (2)Biochemistry, and (3)Oncology, University of Rochester, Rochester, New York 14642

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The C-C chemokines are major mediators of chemotaxis of monocytes and some T cells in inflammatory reactions. The pathways by which the C-C chemokine receptors activate phospholipase C (PLC) were investigated in cotransfected COS-7 cells. The C-C chemokine receptor-1 (CKR-1), the MCP-1 receptor-A (MCP-1Ra), and MCP-1Rb can reconstitute ligand-induced accumulation of inositol phosphates with PLC beta2 in a pertussis toxin-sensitive manner, presumably through Gbeta released from the G(i) proteins. However, these three receptors demonstrated different specificity in coupling to the alpha subunits of the G(q) class. While none of the receptors can couple to Galphaq/11, MCP-1Rb can couple to both Galpha14 and Galpha16, but its splicing variant, MCP-1Rb, cannot. Since MCP-1Ra and -b differ only in their C-terminal intracellular domains, the C-terminal ends of MCP-1Rs determine G protein coupling specificity. CKR-1 can couple to Galpha14 but not to Galpha16, suggesting some of the C-C chemokine receptors, unlike the C-X-C chemokine receptors, discriminate against Galpha16, a hematopoietic-specific Galpha subunit. The intriguing specificity in coupling of the G(q) class of G proteins implies that the chemokines may be involved in some distinct functions in vivo. The commonality of the chemokine receptors in coupling to the G(i)-Gbeta-PLC beta2 pathway provides a potential target for developing broad spectrum anti-inflammatory drugs.


INTRODUCTION

Chemokines are a large family of small (8-10 kDa), inducible, secreted, proinflammatory cytokines, which are produced by various cell types. Members of the chemokine family share 20-90% homology in their amino acid sequences. The sequences usually have four conserved cysteine residues except lymphotactin. On the basis of the positions of the cysteine residues, the chemokine family can be divided into three subfamilies: the C-X-C or alpha family, the C-C or beta family, and the C or family. The alpha family includes IL-8, (^1)GRO (growth-related oncogene), NAP-2, ENA-78, platelet factor 4, IP-10, and GCP-2, while the beta family includes macrophage chemotactic protein (MCP)-1, -2, and -3, RANTES (regulated upon activation, normal T cell expressed and secreted), macrophage inflammatory protein (MIP)-1alpha and -1beta, I309, and C10 (for reviews see (1, 2, 3) ). The newly discovered family has only one member, lymphotactin. Lymphotactin, unlike other chemokines, has only two conserved cysteine residues(4) . The exact physiological and pathophysiological functions of these factors are not yet clearly defined; however, it is generally believed that their main function is recruitment and activation of leukocytes at the site of inflammation.

Two receptors, IL-8RA and IL-8RB, have been cloned for the C-X-C chemokine family(5) . We have characterized the G protein-coupled pathways for these two receptors by using the cotransfection assay(6) . Recently, three receptors for the C-C chemokines were also cloned: CKR-1(7, 8) , MCP-1Ra, and MCP-1Rb(9) . CKR-1 binds to MIP-1alpha, RANTES, MIP-1beta, and MCP-1 with varying affinities. However, only MIP-1alpha and RANTES can induce biological effects at physiological concentrations(7) . MCP-1Ra and -b are two alternative splicing variants, and they differ only in their C-terminal intracellular domains(9) . MCP-1Rb binds to MCP-1 and MCP-3 but not to MIPs, RANTES, or MCP-2(9, 10) . The C-C chemokine receptors share about 50% sequence homology among themselves and less than 30% homology with the C-X-C chemokine receptors(2) .

CKR-1 and MCP receptors have typical structural characteristics of G protein-coupled receptors, and they induce cytosolic Ca efflux(7, 9) , presumably through activation of phospholipase C (PLC). Five cDNAs that encode the alpha subunits of the G(q) class have been characterized, Galpha(q), Galpha11, Galpha14, Galpha15, and Galpha16(11) , all of which can activate all isoforms of PLC beta, PLC beta1-4, to stimulate the release of inositol phosphates (IPs) (12, 13, 14, 15, 16, 17) . COS-7 cells contain Galpha(q) and Galpha11 but not Galpha14, Galpha15, or Galpha16(15) . The expression of Galpha15 and Galpha16 was detected only in hematopoietic cells (Galpha15 may be the mouse counterpart of human Galpha16)(18, 19, 20) , while Galpha14 is expressed in some lineage of hematopoietic cells as well as other cell types(19) . Many receptors, including the IL-8 receptors, were found to couple to some of the alpha subunits of the G(q) class to activate PLC. Recently, the Gbeta subunits of G proteins were also found to activate specific isoforms of PLC beta. The Gbeta-linked pathway may account for the PTX-sensitive activation of PLC mediated by the IL-8 receptors in mature leukocytes(6) .

Since the C-C chemokines play important roles in chemotaxis of monocytes and some T cells, we characterized the G protein-coupled signal transduction pathways for the three C-C chemokine receptors by the cotransfection assay system in COS-7 cells. We found that the C-C chemokine receptors showed different specificity in coupling to the Galpha subunits of the G(q) class, while the receptors can all couple to the G(i) proteins to activate PLC beta2 through Gbeta.


EXPERIMENTAL PROCEDURES

Cell Culture, Transfection, and Assay

COS-7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. For transfection, COS-7 cells (5 times 10^4 cells/well) were seeded in 24-well plates the day before transfection. Plasmid DNAs (0.5 µg/well) were premixed with 1.7 µl of Lipofectamine (Life Technologies, Inc.) and added to cells. The cells were then labeled with [^3H]inositol (5 µCi/ml) 24 h later. The next day, ligand-induced accumulation of IPs was determined as described in (6) . In brief, cells were lysed in 10% perchloric acid and neutralized with KOH. IPs were retained by AG1-X8 ion exchange resin and eluted with formic acid. Portions of eluted samples were counted in a scintillation counter. The basal IP level in COS-7 cells is about 2000 dpm. Transfection or cotransfection with cDNAs encoding the C-C chemokine receptors, Galpha14, PLC beta1, Lac Z, did not alter the basal level. Cells transfected with the Galpha16 and PLC beta2 cDNA increased the basal levels to 2500 and 3500 dpm, respectively. MCP-1 and MIP-1alpha were purchased from R& Systems. All the assays were repeated at least three times. The representative ones were shown.

cDNA Cloning

The cDNAs encoding CKR-1, MCP-1Ra, and MCP-1Rb were cloned from human THP-1 monocytic cells by polymerase chain reaction using primers based on the published sequences(7, 9) . The sequences were verified by DNA sequencing. The cloned receptors were ligated into pcDNA/AMP (Invitrogen).

Ligand Binding Assay

Cos-7 transfectants were incubated with I-labeled ligands (Amersham Corp., 2000 Ci/mmol) in Dulbecco's modified Eagle's medium containing 0.25% bovine serum albumin on ice for 1 h, and the cells were washed by phosphate-buffered saline containing 0.25% bovine serum albumin three times. Finally the cells were solubilized in 0.1 N NaOH, and aliquots were counted by a -counter. The maximum binding sites and affinities were determined by Scatchard analyses.


RESULTS AND DISCUSSION

We tested in cotransfected COS-7 cells whether the newly cloned C-C chemokine receptors, including the MCP-1Ra, MCP-1Rb, and CKR-1, can couple to the alpha subunits of the G(q) class of G proteins. We have previously shown that receptors that can couple to Galpha(q) or Galpha11 gave ligand-induced accumulation of IPs in COS-7 cells transfected with the receptor cDNAs(21) . Thus, to test whether these C-C chemokine receptors can couple to Galpha(q) or Galpha11, we transfected the cDNAs corresponding to each of the C-C chemokine receptors into COS-7 cells and determined ligand-induced accumulation of IPs. There was little MIP-1alpha-induced accumulation of IPs in cells expressing CKR-1, and neither was there MCP-1-induced accumulation of IPs in cells expressing MCP-1Ra or MCP-1Rb (Fig. 1A). These results indicate that these receptors cannot couple to endogenous Galpha(q) or Galpha11. To test whether the receptors can couple to other members of the G(q) class, we cotransfected COS-7 cells with each of the receptor cDNAs and the cDNA corresponding to Galpha14 or Galpha16. We and others have previously found that all the receptors tested in the cotransfection assay, including alpha1A, -B, and -C(21) , beta2-adrenergic receptors(22) , the m2-muscarinic receptor, D1-dopamine receptor, V2,V1a-vasopressin receptor, A2a-adenosine receptor, µ-opioid receptor, 5-1a, 1c/2c serotonin receptors, and thrombin receptor (17) can couple to Galpha16. However, neither CKR-1 nor MCP-1Ra can couple to Galpha16, since cells coexpressing Galpha16 and CKR-1 or MCP-1Ra showed little ligand-induced accumulation of IPs (Fig. 1A). Interestingly, MCP-1Rb, the alternative splicing variant of MCP-1Ra, gave ligand-dependent release of IPs when coexpressed with Galpha16 (Fig. 1A), suggesting that MCP-1Rb can couple to Galpha16. The activation of Galpha16 by MCP-1Rb was insensitive to PTX (Fig. 1B). Furthermore, these C-C chemokine receptors demonstrated different selectivity in coupling to Galpha14; CKR-1 and MCP-1Rb can couple to Galpha14, while MCP-1Ra cannot (Fig. 1A). The concentration-dependent responses to ligand indicate a mean effective concentration (EC) for MCP-1Rb-mediated activation of Galpha16 and Galpha14 of about 7 nM.


Figure 1: Coupling of the C-C chemokine receptors to the alpha subunits of the G(q) class. A, COS-7 cells were cotransfected with the cDNA (0.25 µg) encoding the C-C chemokine receptors and the cDNA (0.25 µg) corresponding to Galpha14, Galpha16, or Lac Z (beta-galactosidase, as a control) as indicated in the figure. Forty-eight hours after transfection, MIP-1alpha-induced (7 nM) accumulation of IPs in cells expressing CKR-1 and MCP-1-induced (20 nM) accumulation of IPs in cells expressing MCP-1Ra or -B were determined 30 min after addition of ligands. B, concentration-dependent accumulation of IPs to MCP-1 was determined in cells coexpressing MCP-1Rb and Galpha14 or Galpha16 in the presence (closed symbols) or absence (open symbols) of PTX. PTX (500 ng/ml) was added 4 h before the PLC assay. C and D, COS-7 cells were cotransfected with the Galpha16 cDNA (0.25 µg) and cDNA (0.25 µg) encoding one of the C-C chemokine receptors. The cells were lysed in SDS sample buffer 48 h after transfection. The proteins were separated on 12% SDS-polyacrylamide gel and electroblotted onto a nitrocellulose membrane. The Galpha16 (C) and Galpha14 (D) proteins were detected with antibodies specific to Galpha16 and Galpha14, respectively.



We have demonstrated in many of our reports (6, 15, 21, 23) that coexpression of one protein does not significantly affect the expression of others. Nonetheless, we determined the expression of Galpha16 and Galpha14 in cells cotransfected with cDNA encoding CKR-1, MCP-1Ra, or MCP-1Rb to eliminate the possibility that the inabilities of CKR-1 and MCP-1Ra to couple to Galpha16 or Galpha14 were the results of lower expression levels of the proteins. As shown by Fig. 1, C and D, the expression levels of Galpha16 and Galpha14 were similar regardless of the nature of the coexpressed receptors. We also determine the receptor levels by using I-labeled MCP-1 or MIP-1alpha. The cells transfected with CKR-1, MCP-1Ra, or MCP-1Rb all show about 525-650 fmol of ligand-binding sites/1 times 10^5 cells, and the affinities are around 4.5 nM for CKR-1, 2.2 nM for MCP-1Ra, and 1.7 nM for MCP-1Rb. Therefore, the inabilities of CKR-1 and MCP-1Ra to couple to Galpha16 or Galpha14 are not due to variations in expression levels.

The responses to the C-C chemokines (including MCP-1, MIP-1alpha, and RANTES) in monocytic phagocytes were found to be mostly PTX-sensitive (1, 2, 3) , yet the signal transduction pathways mediated by the alpha subunits of the G(q) class are PTX-resistant(11) . PTX is a bacterial toxin, which modifies the C-terminal Cys residues of the Galpha(i) and Galpha(o) subunits. The modification prevents interactions between receptors and G proteins. Recently, we proposed a novel pathway to explain the PTX sensitivity; receptors interact with PTX-sensitive G proteins to release the Gbeta subunits, which then activate PLC beta2(6, 23) . Since there are abundant G(i) proteins (predominantly G(i)2 as well as some G(i)3) (24, 25) and the PLC beta2 proteins in leukocytes(26) , the G(i)-Gbeta-PLC beta2 pathway is likely to occur in vivo. To test whether the C-C chemokine receptors can couple to endogenous PTX-sensitive G proteins of COS-7 cells to activate PLC beta2, we transfected COS-7 cells with cDNA encoding PLC beta2 and cDNA encoding each of the C-C chemokine receptors. COS-7 cells contain endogenous Galpha(i)2 but not Galpha(o) proteins, and they contain endogenous PLC beta1 but not PLC beta2 as determined by specific antibodies(14) . The accumulation of IPs in response to varying concentrations of MCP-1 or MIP-1alpha was determined. All three receptors can induce activation of PLC beta2 with EC of 0.5 nM for CKR-1 and 3 nM for MCP-1Ra and MCP-1Rb (Fig. 2). In addition, we found that the ligand-induced responses in cells coexpressing the receptors and PLC beta2 were PTX-sensitive (Fig. 2). Thus, we conclude that all three C-C chemokine receptors can couple to endogenous PTX-sensitive G proteins, presumably the G(i)2 protein, to activate PLC beta2 via Gbeta (the Galpha(i) subunits cannot directly activate PLC beta(14) ). The finding that MCP receptors can inhibit adenylyl cyclase activity in A293 human kidney cells expressing the receptor confirms our notion that the receptor can couple to the G(i) proteins(27) . The inability of the chemokine receptors to activate endogenous PLC beta (Fig. 1A) or recombinant PLC beta1 (data not shown) is consistent with our previous observation that Gbeta could not activate PLC beta1 in the cotransfection system(6, 21) . In addition, we found that MCP-1 could not activate PLC in cells expressing CKR-1 and that MIP-1alpha could not induce IP formation in cells expressing MCP receptors.


Figure 2: Activation of PLC beta2 by the C-C chemokine receptor in transfected COS-7 cells. COS-7 cells were cotransfected with the PLC beta2 cDNA (0.25 µg) and cDNA (0.25 µg) corresponding to CKR-1 (A), MCP-1Ra (B, squares), and MCP-1Rb (B, triangles). Ligand-induced accumulation of IPs was determined 30 min after addition of ligands (MIP-1alpha in A, MCP-1 in B) in the presence (closed symbols) or absence (open symbols) of PTX. PTX (500 ng/ml) was added 4 h before the PLC assay.



Although the C-C chemokine receptors can couple to the G(i)-Gbeta-PLC beta2 pathway, these receptors demonstrate interesting specificity in coupling to the alpha subunits of the G(q) class. While none of the three receptors couples to Galpha(q)/11, MCP-1Rb can couple to both Galpha16 and Galpha14, but its splicing variant MCP-1Ra cannot couple to either Galpha14 or Galpha16. CKR-1 couples to Galpha14 but not to Galpha16. The differences between MCP-1Ra and MCP-1Rb in G protein coupling indicate that the C-terminal intracellular domains are critical in determining the G protein coupling specificity, since these two receptors differ only in the C-terminal ends(9) . Moreover, the finding further supports our previous notion, drawn from our study of the alpha1B-adrenergic receptor, that different receptor sequences are required for activation of different Galpha subunits of the G(q) class(28) . The study of the alpha1B-adrenergic receptor indicates that the alpha1B-adrenergic sequences required for activation of Galpha14 are located in the third intracellular loop, whereas the sequences required for activation of Galpha16 do not appear to be localized within the third inner loop. This report, however, points out that the sequences in the C-terminal intracellular domain are critical for activation of both Galpha14 and Galpha16. We interpret the apparent discrepancy to suggest that G protein-interacting sequences on different receptors may be located at different sites or that there exist multiple G protein-interacting sites on a receptor so that alteration of any one of them abolishes the ability of the receptor to couple to the G protein. In this report we did not account for the influences of different Gbeta subunits on the coupling of these receptors to different Galpha subunits because there were no significant differences observed for different Gbeta subunits in interaction with Galpha(i)2 or in regulation of PLC beta2 (15) or of adenylyl cyclase activities(29) . Furthermore, the same system (COS-7 cells) was used in the studies; thus, the differences in the coupling of these chemokine receptors to different Galpha subunits cannot be attributed to Gbeta.

The physiological relevance of the pathways mediated by Galpha(i), Galpha16, and Galpha14 is not clear. All these Galpha subunits were found in various hematopoietic cells. Although more systematic studies of the expression of these Galpha subunits are needed, previous studies suggest that there are very abundant Galpha(i) subunits with the majority of Galpha(i)2 and some Galpha(i)3 in leukocytes (24, 25) and that the levels of the Galpha(i) subunits increase along with differentiation(18) . Galpha16 and PLC beta2 was detected only in hematopoietic cells. Galpha16 was detected in neutrophils, monocytes, lymphocytes, and erythrocytes as well as various hematopoietic progenitor cells, and its expression in HL-60 promyeloid cells decreases by 90% after differentiation(18) . These results, in addition to the findings that responses to chemokines in mature leukocytes were mostly PTX-sensitive, suggest that the G(i)-linked pathway may be the predominant one in chemokine-mediated effects in mature leukocytes, such as chemotaxis and activation of leukocytes. If this hypothesis is correct, the activation of PLC beta2 by Gbeta would be an excellent target for developing broad spectrum anti-inflammatory drugs, because all the known chemokine receptors, including the C-C chemokine receptors, can couple to the G(i)-Gbeta-PLC beta2 pathway. The fact that PLC beta2 is expressed only in hematopoietic cells may limit potential side effects.

Recently, some evidence indicates that chemokines may be directly or indirectly involved in the regulation of hematopoiesis; MIP-1alpha inhibits proliferation of the hematopoietic stem cells(30) , and the IL-8 receptor-null mice have expanded populations of neutrophils and B cells, in addition to their reduced abilities to respond to inflammatory stimuli(31) . The Galpha16-linked pathway may play a role in hematopoiesis as well as in other hematopoietic functions, although there is a lack of evidence. Nevertheless, regulation of expression levels by differentiation, specificity in interactions between receptors and G proteins and between G proteins and effectors, and diversity of molecular natures of receptors, G proteins, and effectors in leukocytes underlie the molecular basis for the complex function of signal transduction networks in the hematopoietic system. Alternative splicing further expands the signal-processing capabilities of eukaryotic cells.


FOOTNOTES

*
This work was supported by the Pharmaceutical Research and Manufacturers Foundation of America, Inc. and a National Institutes of Health Grant GM53162R29 (to D. W.), by the Leukemia Society of America, and by Grant IRG-18 from the American Cancer Society (to H. J.). 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.

§
To whom correspondence should be addressed. Tel.: 716-275-2029; Fax: 716-244-9283.

(^1)
The abbreviations used are: IL, interleukin; MCP, macrophage chemotactic protein; RANTES, regulated upon activation, normal T cell expressed and secreted; MIP, macrophage inflammatory protein; PLC, phospholipase C; IP, inositol phosphate; PTX, pertussis toxin.


REFERENCES

  1. Oppenheim, J. J., Zachariae, O. C., Mukaida, N., and Matsushima, K. (1991) Annu. Rev. Immunol. 9, 617-648 [CrossRef][Medline] [Order article via Infotrieve]
  2. Murphy, P. M. (1994) Annu. Rev. Immunol. 12, 593-633 [CrossRef][Medline] [Order article via Infotrieve]
  3. Schall, T. J. (1994) in The Cytokine Handbook (Thomson, A., ed) pp. 419-460, Academic Press, New York
  4. Kelner, G. S., Kennedy, J., Bacon, K., Jenkins, N. A., Bazan, J., Moore, K., Schall, T. J., and Zlotnik, A. (1994) Science 266, 1395-1400 [Medline] [Order article via Infotrieve]
  5. Murphy, P. M., and Tiffany, H. L. (1991) Science 253, 1280-1283 [Medline] [Order article via Infotrieve]
  6. Wu, D., LaRosa, G. J., and Simon, M. I. (1993) Science 261, 101-103 [Medline] [Order article via Infotrieve]
  7. Neote, K., DiGreorio, D., Mak, J., Horuk, R., and Schall, T. (1993) Cell 72, 415-425 [Medline] [Order article via Infotrieve]
  8. Gao, J. L., Kuhns, D. B., Tiffany, H. L., Li, X., Francke, U., and Murphy, P. M. (1993) J. Exp. Med. 177, 1421-1427 [Abstract]
  9. Charo, I. F., Myers, S. J., Herman, A., Franci, C., Connolly, A. J., and Coughlin, S. R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2752-2756 [Abstract]
  10. Franci, C., Wong, L. M., Damme, J., Proost, P., and Charo, I. F. (1995) J. Immunol. 156, 6511-6517
  11. Simon, M. I., Strathman, M. P., and Gautum, M. (1991) Science 252, 802-808 [Medline] [Order article via Infotrieve]
  12. Smrcka, A., Hepler, J., Brown, K., and Sternweis, P. (1991) Science 251, 804-807 [Medline] [Order article via Infotrieve]
  13. Taylor, S., Chae, H., Rhee, S., and Exton, J. (1991) Nature 350, 516-518 [CrossRef][Medline] [Order article via Infotrieve]
  14. Wu, D., Lee, C-H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817 [Abstract/Free Full Text]
  15. Wu, D., Katz, A., and Simon, M. I. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5297-5301 [Abstract]
  16. Lee, C. H., Park, D., Wu, D., Rhee, S.-G., and Simon, M. I. (1992) J. Biol. Chem. 267, 16044-16047 [Abstract/Free Full Text]
  17. Jiang, H.-J., Wu, D., and Simon, M. I. (1994) J. Biol. Chem. 269, 7593-7596 [Abstract/Free Full Text]
  18. Amatruda, T., Steele, D. A., Slepak, V. Z., and Simon, M. I. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5587-5591 [Abstract]
  19. Wilkie, T., Acherle, P. A., Strathmann, M. P., Slepak, V. Z., and Simon, M. I. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10049-10053 [Free Full Text]
  20. Wilkie, T., Gilbert, D. J., Olsen, A. S., Chen, X. N., Amatruda, T., Kroenberg, J. R., Track, B. J., de Jong, P., Reed, R. R., Simon, M. I., Jenkins, N. A., and Copeland, N. G. (1992) Nat. Genet. 1, 85-91 [Medline] [Order article via Infotrieve]
  21. Wu, D., Katz, A., Lee, C.-H., Jiang, H., and Simon, M. I. (1992) J. Biol. Chem. 267, 25798-25802 [Abstract/Free Full Text]
  22. Wu, D., Kuang, Y., Wu, Y., and Jiang, H. (1995) J. Biol. Chem. 270, 16008-16010 [Abstract/Free Full Text]
  23. Katz, A., Wu, D., and Simon, M. I. (1992) Nature 360, 686-689 [CrossRef][Medline] [Order article via Infotrieve]
  24. Murphy, P. M., Eide, B., Goldsmith, P., Brann, M., Gierschik, P., Spiegel, A. M., and Malech, M. L. (1987) FEBS Lett. 221, 81-86 [CrossRef][Medline] [Order article via Infotrieve]
  25. Goldsmith, P. K., Rossiter, K., Carter, A., Simond, W., Unson, C. G., Vinitsky, R., and Spiegel, A. M. (1988) J. Biol. Chem. 263, 6476-6479 [Abstract/Free Full Text]
  26. Kriz, R., Lin, L.-L., Ellist, C., Heldin, C.-H., Pawson, T., and Knopf, J. (1990) CIBA Found. Symp. 150, 112-117 [Medline] [Order article via Infotrieve]
  27. Myers, S. J., Wong, L. M., and Charo, I. F. (1995) J. Biol. Chem. 270, 5786-5792 [Abstract/Free Full Text]
  28. Wu, D., Jiang, H., and Simon, M. I. (1995) J. Biol. Chem. 270, 9828-9832 [Abstract/Free Full Text]
  29. Ueda, N., Lee, E., Smrcka, A. V., Robishaw, J. D., and Gilman, A. G. (1994) J. Biol. Chem. 269, 4388-4395 [Abstract/Free Full Text]
  30. Graham, G., Wright, E. G., Hewick, R., Wolpe, S., Donalson, D., Lorimore, S., and Pragnell, I. B. (1990) Nature 344, 442-445 [CrossRef][Medline] [Order article via Infotrieve]
  31. Cacalano, G., Lee, J., Ryan, A., Meek, S., Hultgren, B., Wood, W., and Moore, M. W. (1994) Science 265, 682-684 [Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.