Immunology Letters, 26 (1990) 1-6
Elsevier IMLET 01486
GTP binding proteins and signal transduction in the human neutrophil Ronald Weingarten and Gary M. Bokoch Department of Immunology, Research Institute of Scripps Clinic, La Jolla, CA, U.S.A.
(Received24 May 1990; accepted2 July 1990)
1. Introduction Neutrophils play a major role in the body's ability to defend against bacterial infection, as evidenced by the often life-threatening infections acquired by patients with neutropenia, leukemia or congenital diseases affecting neutrophil structure and function. Neutrophils utilize a highly developed chemotactic sensing system to mobilize in response to microbial infection, and they are able to destroy microorganisms with an array of microbicial oxidants, proteolytic enzymes and antimicrobial peptides. Under certain circumstances, the excessive or inappropriate release of these highly destructive agents can also result in undesirable tissue damage and inflammation. Neutrophils can be considered the primary cellular mediators of inflammation and have been implicated in the pathogenesis of diseases such as rheumatoid arthritis, adult respiratory distress syndrome and myocardial infarction. It is clear that a rational approach to modulation or therapeutic intervention in such disease states will require an understanding of the biochemical mechanisms through which the neutrophil responds to bacterial and physiological chemoattractant factors. The activation of neutrophils by various chemoattractant factors is initiated by binding of these ligands to specific cell surface receptor proteins. Upon ligand binding, the receptor is "activated" and a signal is transferred to the cell interior which
Correspondence to: RonaldWeingarten,M.D.,Dept. of Immunology, Research Institute of Scripps Clinic, 10660 N. Torrey Pines Road IMM-12,La Jolla, CA 92037, U.S.A.
results in initiation of the complex cellular responses to the chemoattractant stimulus. Substantial evidence indicates the involvement of a family of proteins known as GTP binding regulatory proteins as key components in transducing the signal produced upon receptor activation into a form that is recognizable by various cellular effector systems.
2. GTP binding regulatory proteins: history and definitions The hormone-regulated adenylate cyclase system has been exceptionally useful for the study of cellular transmembrane signaling. While this system was originally shown to be composed of receptors and adenylate cyclase, several observations led to the hypothesis that an intermediary element was interposed between these two components. In 1971, Rodbell and colleagues  demonstrated that hormonal activation of adenylyl cyclase required guanosine triphosphate (GTP). Subsequently, Maguire and coworkers  found that guanine nucleotides affected the affinity of agonists, but not antagonists, for receptor. The demonstration by Cassel and Selinger  that ~-adrenergic agonists stimulate GTP hydrolysis further supported the hypothesis that guanine nucleotides act on a regulatory site in the adenylate cyclase system. This site was identified as a distinct guanine nucleotide binding protein or G protein in a series of landmark experiments by Gilman, Ross and colleagues. These experiments included purification of the G protein, Gs, and its functional reconstitution into plasma membranes from a mutant cell line devoid of G s [4-8]. Subsequent studies have shown that G proteins
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specifically bind and become activated by guanine nucleotides, particularly GTP. Energy derived from the binding and hydrolysis of GTP is utilized by the G protein to couple membrane receptors to cellular effectors. For G protein-regulated transmembrane signaling systems, the following model has evolved (Fig. 1): an agonist (e.g., a hormone, neurotransmitter or autacoid) binds to its specific receptor, thus promoting the interaction of receptor with G protein. As a result of this interaction, the G protein changes its conformation and releases tightly bound guanosine diphosphate (GDP). The empty guanine nucleotide binding site on the G protein created by GDP release is immediately filled by the more abundant guanosine triphosphate (GTP). GTP binding causes dissociation of the G protein a subunit from the ~/3' subunit. This GTP-bound form of the G protein o~ subunit can activate intracellular effector molecules (such as adenylyl cyclase), thereby modulating cell function. In order to terminate effector activation, the GTP is hydrolyzed to GDP by a GTPase intrinsic to the G protein o~ subunit. A growing number of highly homologous, largely membrane-associated proteins have been identified as members of the receptor-coupled G protein family (Fig. 2, Family I; reviewed in refs. 9 and 10). These proteins share a heterotrimeric structure, consisting of distinct o~ and similar ~/7 subunits. Specific G protein oligomers were originally named on the basis of function; e.g., G s stimulates adenylyl cyclase and G i inhibits adenylyl cyclase. However, a multitude of newly discovered G proteins [11, 12] in combination with multiple functions being ascribed to known proteins have made this system of nomenclature confusing and inadequate to deal with the current level of G protein complexity. Future nomenclature may reflect the precise primary amino acid sequence of the various G protein subunits. G protein ~ subunits range in size from 39 to 52 kDa and contain the binding sites for guanine nucleotides. Many of the G protein ~ subunits demonstrate a marked degree of sequence homology, particularly in their putative guanine nucleotide binding sites, as well as in regions that are postulated to involve receptor and/or subunit interactions [9, 10]. The a subunits contain sites available for chemical modification by various bacterial toxins, resulting in either stimulation or inhibition of the activity of the G protein. Known especially as ADP ribosyla-
tion, this is an important mechanism by which bacteria modulate the cellular signal transduction machinery of their host. 3. Evidence for the involvement of G proteins in transduction of chemoattractant receptor signaling The evidence that GTP binding proteins couple neutrophil receptors to their effector systems is based on a number of experimental observations (reviewed in refs. 13-16). Many of these observations pertain to the neutrophil N-formyl peptide chemoattractant receptor. This receptor binds Nformyl peptides which are thought to represent breakdown products of secreted prokaryotic proteins, and which are potent activators of neutrophil function. The N-formyl peptide receptor of neutrophils exists in two states, with relatively high vs. low affinities for binding N-formyl peptides [17-19]. These forms of the receptor do not represent distinct isoforms of the N-formyl peptide receptor, but rather reflect the presence of the "free" receptor (low affinity form) vs. the form that is coupled to a GTP binding protein (high affinity form), as evidenced by the rapid and complete interconvertibility of these forms by guanine nucleotides (Fig. 1). The demonstration that ligand binding to the N-formyl peptide receptor results in both stimulation of membrane guanine nucleotide binding and GTP hydrolytic activity is further indication of receptor coupling to GTP binding proteins. The utilization of pertussis toxin has implicated G proteins in the signal transduction process of the neutrophil. The toxin prevents neutrophil activation via N-formyl peptides by catalyzing the ADP ribosylation of a 40-kDa G protein t~ subunit in neutrophil membranes [20, 21]. This covalent modification of the G protein results in functional uncoupling of the chemoattractant receptor from its G protein transduction partner. Finally, an enzyme critical to neutrophil function, phospholipase C, has been the subject of additional investigations supporting G protein involvement in signal transduction processes. It has been found that GTP is a requirement for effective activation of phospholipase C by the N-formyl peptide receptor in membrane preparations and that nonhydrolyzable guanine nucleotides or other G protein
Fig. 1. Activation-deactivation cycle of the receptor-coupled G proteins. Boxes with a "V-shaped" indentation represent the lowaffinity form of receptor; those with a "rectangular" indentation represent the high-affinity form. L, ligand/agonist; R, receptor; G, G protein; c~,/~, 7, G protein subunits; E, effector.
activators can directly stimulate phospholipase C in such preparations . These findings imply that a GTP binding protein directly couples the chemoattractant receptor to phospholipase C. The aforementioned observations apply not only to activation via the N-formyl peptide receptor, but have been extended to activation by leukotriene B4, C5a, platelet activating factor, and IgGs . While a pertussis toxin-sensitive GTP binding protein has been clearly implicated in coupling chemoattractant receptors to neutrophil activation via phospholipase C, roles for additional GTP binding proteins in the activation process have not been ruled out. Indeed, recent data has demonstrated that guanine nucleotides stimulate neutrophil degranulation and oxidant production (via the NADPH oxidase) by pertussis toxin-insensitive pathways. This suggests the involvement of non-pertussis toxinsensitive GTP binding proteins in these processes . This possibility has been made even more likely by our recent identification of a number of previously uncharacterized, non-pertussis toxin substrate GTP binding proteins in the human neutrophil, as discussed in the following section.
4. The GTP binding protein composition of human neutrophils In our studies of neutrophil signal transduction, we have taken the initial approach of identifying, purifying, and characterizing the GTP binding pro-
teins present in human neutrophils. These cells contain both a "classical" form of GTP binding protein which serves as the major pertussis toxin substrate of human neutrophils, and, additionally, a group of low-molecular-weight GTP binding proteins which exhibit distinctly different physical and functional characteristics (Fig. 2, Family II). The major pertussis toxin substrate of mature human neutrophils has a 40-kDa ~ subunit. When isolated in the absence of G protein activators, it purifies as a complex with /~/7 subunits and can be shown to interact functionally with purified /3/7 subunits from several sources [23, 24]. This protein, termed G n, thus exhibits a typical G protein od/t/7 subunit structure. Purified G n can be stoichiometrically ADP ribosylated by pertussis toxin. Proteolytic mapping and immunological characterization in our laboratory , as well as others [23, 24, 26], indicate G n is likely to be identical to the G protein known as Gi-2, the gene for which was originally cloned by Itoh et al. . Studies on the subcellular distribution of G n indicate that in addition to the plasma membrane, a cytoplasmic form of G n also occurs in the neutrophil [25, 28]. This soluble G n exists as the oL subunit uncomplexed from ~/7 subunits. The processes that might regulate the membrane vs. cytosolic distribution of G n are as yet undefined, but may relate to both the availability of ~/3' subunits to anchor G not to the membrane, as well as to the presence of covalently attached fatty acids on the G n ot subunit. In addition to G n, we have purified two GTP binding proteins of 24 and 26 kDa, termed G24K and G26K. These proteins exhibit the property of binding guanine nucleotides after exposure to denaturing conditions (i.e., SDS-PAGE, nitrocellulose transfer), a property not shared by G n or other "classical" G proteins. This binding is of high affinity and is specific for guanine nucleotides. A third low-molecular-weight GTP binding protein termed G22Kwas also isolated, which binds guanine nucleotides only under native conditions. Immunological studies in our laboratory have demonstrated that G22 K and G26K are unrelated to Ga or the classical G proteins and are thus not proteolytic breakdown products of these proteins. Additionally, none of the G22K-G26K proteins is a substitute for pertussis or cholera toxins.
The Receptor-Coupled (Oligomeric) G Proteins (MW = 39 kD-52 kD)
The Low Molecular Weight 13 Proteins (MW = 19 kD-28 kD)
Gs, Gi, Go, Gz, T n
Ras, rho, ral, rab, rap, rac, stag-p25
afJy Subunit Structure
Single Known Subunit
a/[J Adrenergic, Muscarinic, Rhodopsin, fMLP, many others
Adenylyl Cyclase, Ca ++ Channels, c6MP Phosphodiesterase, others
? 6TPase Activating Protein
Cholera and/or Pertussis Toxin
Fig. 2. Characteristics of the members of the G protein superfamily.
Subsequent work has shown that the G22 K protein can actually be resolved into two 22-kDa GTP binding proteins. One of these proteins, which has been tentatively identified as a form of rac , is a substrate for the botulinum toxin C a ADPribosyltransferase. Additional C 3 substrates, the rho proteins, have been identified and are present in human neutrophil cytosol . The normal cellular roles of the botulinum Ca substrates and whether these GTP binding proteins participate in the pathophysiology of botulism is unknown. Use of the C a enzyme will hopefully serve as a tool to delineate some of the physiological roles of rac (and rho) in the neutrophil, as well as in other cell types. The major component of G22K is a form of rap 1 [31, 32]. We have shown rap 1 to bind guanine nucleotides in a manner which is regulated by the presence of endogenous G D P and which can be regulated by Mg 2+ ion . Rap 1 is also an excellent substrate for cAMP-dependent protein kinase, both in vitro and in vivo, and may participate in the inhibition of neutrophil activation by agents able to 4
increase neutrophil cAMP levels . Data which suggest that rap 1 may be able to interact with the cytochrome b component of the N A D P H oxidasesuperoxide generating system has also been reported I34]. Rap 1 has structural similarities to the ras family of GTP binding proteins. The ras proteins are a group of cancer-associated gene (proto-oncogene) products found in over 30°7o o f all human cancers. The rap 1 protein, also referred to as Krev 1, was reported to reverse K-ras induced transformation of mouse fibroblasts . Its activity as a potential anti-oncogene is currently a major subject of investigation. Interactions of rap (or rac) with receptors involved in neutrophil differentiation or cell priming, such as those for granulocyte-macrophage colony stimulating factor (GM-CSF) or bacterial lipopolysaccharides (LPS), analogous to the interactions of ras with growth factor receptors, are possibilities.
5. G proteins and signal transduction in the human neutrophil Since G n is the major pertussis toxin substrate in the human neutrophil, it is a likely candidate for coupling chemotactic factor receptors to phospholipase C in the neutrophil. Hopefully, the availability of purified protein will allow convincing demonstration of the physical interaction of this G protein with chemoattractant receptor and phospholipase C in vitro. The presence of cytoplasmic G n in the neutrophil might indicate roles for this signal-transducing protein in modulating enzymes or processes not solely localized to the plasma membrane. Alternatively, the signal transduction process itself might be modulated by varying the membrane levels of G not transduction units available to chemotactic factor receptors, with the cytoplasm serving as a pool of transduction units. The discovery of the low-molecular-weight G proteins in the neutrophil is of extreme interest in terms of the transduction process. These proteins are potentially involved in guanine nucleotide-mediated granule exocytosis or oxidative burst activity. Additional roles could involve protein translocation, phospholipase regulation, and cell differentiation and/or priming effects. The additional possibility that these proteins may interact with chemoattractant receptors directly in the neutrophil would be exciting and needs to be examined rigorously. Clearly, a total understanding of the signal transduction process in the neutrophil will require definition of the functional roles of these GTP binding proteins.
those diseases known to affect normal neutrophil function. Studies to further characterize the function of these proteins will undoubtedly improve our understanding of the immune system in general and may enhance our ability to intervene in a multitude of disease states. As the relevance of G protein dysfunction to disease processes becomes more clearly understood, research laboratory assays of G protein levels and activity currently in use may find application in the clinical immunology laboratory. In addition to diagnostic studies, it is likely that increased knowledge of G protein function will result in targeting of G proteins for potential therapeutic intervention. As an example, neutrophils have been implicated in several conditions characterized by destructive, nonpurposeful, inflammatory processes, such as adult respiratory distress syndrome and rheumatoid arthritis. Future experimental approaches to the therapy of such inflammatory disorders may include pharmacologic blockade of G protein-coupled transduction pathways to decrease neutrophil activation and diminish tissue destruction. In the best of circumstances, the potential for drug intervention using G proteins as targets may acquire the scope and success that has resulted from targeting cell surface receptors.
Acknowledgements We thank Monica Bartlett for her helpful suggestions and superb editorial assistance.
References 6. Clinical significance of G proteins in neutrophil function The discovery and characterization of G proteincoupled signal transduction in the human neutrophil has markedly expanded our knowledge of events required for neutrophil activation. The potential clinical significance of these findings should not be underestimated as G protein dysfunction, either on a qualitative or quantitative basis, may have farreaching pathophysiologic importance. Future experimental efforts may implicate G proteins in disease mechanisms, particularly those diseases resulting from aberrant neutrophil function as well as
 Rodbell, M., Birnbaumer, L. and Pohl, S. L. et al. (1971) J. Biol. Chem. 246, 1877.  Maguire, M. E., Van Arsdale, P. M. and Gilman, A. G. (1976) Mol. Pharmacol. 12, 335.  Cassel, D. and Selinger, Z. (1976) Biochim. Biophys. Acta 452, 538.  Ross, E. M. and Gilman, A. G. (1977) Proc. Natl. Acad. Sci. USA 74, 3715.  Ross, E. M. and Gilman, A. G. (1977) J. Biol. Chem. 252, 6966.  Ross, E. M., Howlett, A. C., Ferguson, K. M. et al. (1978) J. Biol. Chem. 253, 6401.  Northup, J. K., Sternweis, P. C., Smigel, M. D. et al. (1980) Proc. Natl. Acad. Sci. USA 77, 6516.  Sternweis, P. C., Northup, J. K., Smigel, M. D. et al. (1981) J. Biol. Chem. 256, 11517.
 Gilman, A. G. (1987) Annu. Rev. Biochem. 56, 615.  Gilman, A. G. (1989) J. Am. Med. Assoc. 262, 1731.  Strathmann, M., Wilkie, T. M. and Simon, M. I. (1989) Proc. Natl. Acad. Sci. USA 86, 7407.  Libert, E, Parmentier, M., Lefort, A., Dinsart, C., Van Sande, J., Maenhaut, C., Simons, M., Dumont, J. E. and Vassart, G. (1989) Science 244, 569.  Omann, G. M., Allen, R. A., Bokoch, G. M., Painter, R. G., Traynor, A. E. and Sklar, L. A. (1987) Physiol. Rev. 67,285.  Snyderman, R., Smith, C. D. and Verghese, M. W. (1986) J. Leukocyte Biol. 40, 785.  Bokoch, G. M. (1989) in: Mechanisms of Leukocyte Activation: Current Topics in Membranes and Transport Vol. 35, (S. Grinstein and O. D. Rothstein, Eds.) pp. 65-101. Academic Press, Orlando, FL.  Sklar, L. A. (1986) Adv. Immunology (F. J. Dixon, Ed.) 39, 95.  Koo, C., Lefkowitz, R. J. and Snyderman, R. (1983) J. Clin. Invest. 72, 748.  Snyderman, R., Pike, M. C., Edge, S. and Lane, B. (1984) J. Cell Biol. 98, 444.  Sklar, L. A., Bokoch, G. M., Button, D. and Smolen, J. E. (1987) J. Biol. Chem. 262, 135.  Bokoch, G. M. and Gilman, A. G. (1984) Cell 39, 301.  Okajima, F. and Ui, M. (1984) J. Biol. Chem. 259, 13863.  Snyderman, R., Smith, C. D. and Verghese, M. W. (1986) J.
Leukocyte Biol. 40, 785.  Oinuma, M., Katada, T. and Ui, M. (1987) J. Biol. Chem. 262, 8347.  Gierschik, P., Sidiropoulous, D., Spiegel, A. and Jakobs, K. H. (1987) Eur. J. Biochem. 165, 185.  Bokoch, G. M., Bickford, K. and Bohl, B. P. (1988) J. Cell. Biol. 106, 1927.  Uhing, K. V., Polakis, P. G. and Snyderman, R. (1987) J. Biol. Chem. 262, 15575.  Itoh, H., Kozasa, T., Nagata, S., Nakamura, S., Katada, T. et al. (1986) Proc. Natl. Acad. Sci. USA 83, 3776.  Rotrosen, D., Gallin, J. I., Spiegel, A. M. and Malech, H. L. (1988) J. Biol. Chem. 263, 10958.  Didsbury, J., Weber, R. F., Bokoch, G. M., Evans, T. and Snyderman, R. (1989) J. Biol. Chem. 264, 16378.  Quilliam, L. A., Lacal, J. and Bokoch, G. M. (1989) FEBS Lett. 247, 221.  Bokoch, G. M., Parkos, C. A. and Mumby, S. M. (1988) J. Biol. Chem. 263, 16744.  Bokoch, G. M. and Quilliam, L. C. (1990) Biochem. J. 267, 407.  Mueller, H. and Sklar, L. A. (1989) J. Cell. Biochem. 40, 27.  Quinn, M.T., Parkos, C.A., Walker, L., Orkin, S. H., Dinauer, M. C. and Jesaitis, A. J. (1989) Nature 342, 198.  Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y. and Noda, M. (1989) Cell 56, 77.