Heterotrimeric G Protein Signaling: Review Roles in Immune Function and Fine-Tuning by RGS Proteins

Home , Heterotrimeric G protein, Rap1, RGS14

Immunity, Vol. 8, 1–10, January, 1998, Copyright 1998 by Cell Press Heterotrimeric G Protein Signaling: Review Roles in Immune Function and Fine-Tuning by RGS Proteins

John H. Kehrl* of a heterotrimeric G protein to release guanosine di- B Cell Molecular Biology Section phosphate (GDP) and to bind guanosine triphosphate

Laboratory of Immunoregulation (GTP) in its place (Figure 1). In the GTP-bound form, G␣ National Institutes of Allergy dissociates from G␤␥, each of which independently binds and Infectious Disease and activates downstream effectors. Signaling is termi-

National Institutes of Health nated when G␣ subunits hydrolyze GTP, returning to the Bethesda, Maryland 20892 GDP-bound state, which results in reassembly with G␤␥ to form the inactive heterotrimers. Besides their intrinsic Immunologists hope to understand how extracellular GTPase activity, several mechanisms suppress down- signals modulate and regulate the function of cells that stream signaling by G␣. Since G␤␥ inhibits guanine nucle- participate in immune responses. Not surprisingly, most otide dissociation, assembly of the heterotrimer inhibits recent signal transduction studies examining immune G␣ nucleotide exchange. In addition, RGS proteins ac- cells have focused on Fc, antigen, and cytokine receptor celerate the slow intrinsic rate of the G␣ GTPase reaction signaling (reviewed by Daeron, 1997; Qian and Weiss, (see below). RGS proteins represent a new class of 1997; O’Shea, 1997, respectively). In these pathways, GTPase activators (GAPs) that function in vivo to modu- tyrosine kinases and phosphatases play dominant roles late signal transduction through GPCRs. as signal transducers. The discovery of the Jak/STAT (Janus kinase/signal transducer and activator of T cells) Heterotrimic G Proteins: Activators pathway as a major mediator of cytokine receptorsignal- of Signaling Pathways ing is an excellent example of their importance. G proteins have been shown to interact directly with However, the recognition of the critical importance only a few effectors: namely, retinal cyclic guanosine of chemokine receptor signaling (reviewed by Murphy, monophosphate phosphodiesterase, several isoforms 1996) is leading to a resurgence of interest in heterotrim- of adenylyl cyclase, phospholipase C (PLC) ␤, and an eric guanine nucleotide–binding proteins (G proteins) as inward rectifying potassium channel. However, a variety signal transducers in immunocompetent cells. Also, the of signaling pathways are activated by the different sub- findings in many cellular systems of significant cross- families of heterotrimeric G proteins (reviewed by Of- talk between signaling pathways provide an impetus fermanns and Simon, 1996). The major cell signaling to examine how signaling through G protein–coupled events and pathways activated by different G subunits receptors (GPCRs) might impinge on signaling through ␣ and their associated G␤␥ subunits are detailed below Fc, antigen, or cytokine receptors. This review focuses and summarized in Table 1. Of note, the majority of on the known and potential roles of heterotrimeric G studies examining heterotrimeric G proteins signaling protein signaling in immune cells and how a new class have been performed with nonlymphoid cells. Although of proteins, termed regulators of G protein signaling there is relatively little information about heterotrimeric (RGS), may function to fine-tune these responses. G protein expression in lymphoid cell subsets, a summary of what is known is provided below.

Heterotrimeric G Proteins: The Players Gs subfamily members stimulate adenylyl cyclases, GPCRs are heptahelical receptors such as the chemo- ubiquitous enzymes that catalyze the conversion of kine receptors that link to downstream signaling path- adenosine triphosphate to the intracellular messenger ways by activating heterotrimeric G proteins. In their cyclic adenosine monophosphate (cAMP) (reviewed by inactive state, heterotrimeric G proteins are composed Gilman, 1995). There are nine isoforms of adenylyl cy- of three subunits, termed ␣, ␤, and ␥ (reviewed by Neer, clase, all of which are stimulated by Gs.Gs␣ can be 1995; Hamm and Gilchrist, 1996; Offermanns and Simon, activated by cholera toxin, which adenosine diphos- 1996). There are 23 distinct ␣ subunits encoded by 17 phate (ADP)–ribosylates an arginine residue, causing the different genes that are divided into four subfamilies irreversible binding of GTP to Gs␣. Cholera toxin or Gs based on primary sequence homology and shared intra- activators increase cAMP levels, which activates cAMP- cellular effector molecules: Gi␣ (Gi␣1-3,Gz␣,Go␣1/2,Gt␣, and dependent protein kinase A (PKA). In endocrine-derived

Ggust␣);Gq␣(Gq␣,G11␣,G14␣, and G15/16␣); Gs␣ (Gs␣ and Golf␣); cells, Gs-mediated signals enhance cell proliferation, and G12␣ (G12␣ and G13␣). The ␤ and ␥ subunits exist either and mutationally activated forms of Gs␣ have been de- as part of the heterotrimeric G protein or together as a tected in some endocrine tumors (reviewed by Spiegel, dimer, but not independently. There are 5 different ␤ 1997). Conversely, activated forms of Gs␣ suppress the subunits and 10 different ␥ subunits, which do not pair transformation of NIH3T3 cells by oncogenic Ras and indiscriminately, since certain ␤ subunits prefer certain inhibit mitogen-activated protein kinase (MAPK) activa- ␥ subunits. By combining different ␣, ␤, and ␥ subunits, tion (Chen and Iyengar, 1994). Thus, the effects of a large number of distinct heterotrimeric G proteins can Gs-mediated signals may have profoundly different ef- be assembled within a cell. fects, depending on the cellular context.

Upon ligand binding, GPCRs stimulate the ␣ subunit Gs␣ is well expressed in most B and T cells and cell lines, although an occasional cell line contains low lev- * To whom correspondence should be addressed (e-mail: jkehrl@ els. For example, the B cell lines Daudi and WEHI231 atlas.niaid.nih.gov). have nearly undetectable levels by immunoblotting (Grant Immunity 2

Figure 1. The GTPase Cycle and Role of RGS Proteins in Limiting the Life Span of GTP-

Bound G␣ Subunits

The activated receptor induces G␣ to exchange GDP for GTP, resulting in dissociation

of theheterotrimer. Both G␤␥ and GTP–G␣ can activate downstream effectors. The RGS proteins enhance the intrinsic GTPase activity of

the G␣ subunit, resulting in reassembly of the heterotrimeric G protein and decreasing the

duration of signaling by the G␣ and G␤␥ subunits.

et al., 1997). Similar to its effects on NIH3T3 cells, Gs- interleukin-2 (IL-2) gene transcription by interfering with mediated signals and subsequent PKA activation inhibit a calcium-sensitive T cell signal transduction pathway lymphocyte proliferation and impair activation of MAPK (Paliogianni et al., 1993). In B lymphocytes it blocks and the related stress-activated protein kinase (SAPK, lipopolysaccharide- and IL-4–induced increases in B also known as Jun kinase) pathways (Tamir et al., 1996). lymphocyte cell size and major histocompatibility com-

The effects of ␤-adrenoceptor–mediated signals (Gs- plex class II expression but enhances IL-4–directed iso- coupled) on lymphocyte function depend on the devel- type switching to IgE (Roper et al., 1994; Fedyk and opmental stage and differentiation status of the cell Phipps, 1996). Increased intracellular levels of cAMP are (reviewed by Sanders, 1995; Scherer et al., 1995). Pros- also implicated in the triggering of apoptosis in thymo- taglandin E2–mediated signals (also Gs-coupled) raise cytes and B lymphocytes. The latter is rescuable by cAMP levels in T cells and reportedly shift the balance either CD2 or CD40 cross-linking (Baixeras et al., 1996). of a cellular immune response toward a T helper type Gi subfamily members inhibit adenylyl cyclase types 2 (Th2) response and away from a Th1 response (re- I, V, and VI and thereby lower cAMP levels (reviewed viewed by Fedyk et al., 1996). Prostaglandin E2 inhibits by Gilman, 1995). Gi␣1-3 and Go␣ are substrates for ADP

Table 1. Signaling by Heterotrimeric G Proteins

Subunit Family Signaling

Gs␣ (1) Activation of adenylyl cyclases

Gi␣ (1) Inhibition of adenylyl cyclases (2) Activation of PI3K␥ (3) Activation of the Ca2ϩ permeable cation channel CD20 (4) Activation of a voltage-independent Ca2ϩ channel in erythroid precursors

Gq␣ (1) Activation of PLC␤ (2) Activation of Btk

G12␣ (1) Increase Naϩ/Hϩ exchange

(2) Increased stress-activated protein kinase activity (G12␣ via a Ras-dependent pathway

and G13␣ via a Ras-independent pathway) (3) Induction of actin polymerization and focal adhesions (Rho dependent) (4) Activation of early response genes and mitogenesis (5) Induction of nitric oxide synthase

G␤␥ (1) Activation of G protein–coupled receptor kinases (2) Activation of PLC␤ (3) Activation of Src family tyrosine kinases

(4) Activation of ion channels (IKG and ICa) (5) Activation of PI3K

See the review by Offermanns and Simon (1996). For G␤␥ activation of PI3K see Stephens et al. (1997). Review 3

Table 2. Results of Gene Targeting Experiments Relevant to G Protein Signaling in Lymphocytes and Inflammatory cells

Molecule Function Phenotype

Gi␣2 G ␣ subunit Increased numbers of single-positive thymic T cells; peripheral T cells that produce high levels of IL-2, interferon-␥, and tumor necrosis factor in response to CD3 cross-linking (Hornquist et al., 1997) SDF-1 Chemokine Major defect in fetal liver and bone marrow B lymphopoiesis (Nagasawa et al., 1996) MIP-1␣ Chemokine No overt abnormality; impaired inflammatory response to influenza virus (Cook et al., 1995) Eotaxin Chemokine Impaired eosinophil recruitment following antigen challenge; decreased peripheral eosinophils (Rothenberg et al., 1997) BLR1 Putative chemokine Lack of inguinal lymph nodes and germinal receptor centers; migration of lymphocytes into splenic follicles is abnormal (Forster et al., 1996) CCR1 Chemokine receptor Impaired neutrophil chemotaxis; no obvious lymphocyte phenotype, although there may be an alteration in the balance between type 1 and type 2 cytokines (Gao et al., 1997) CCR2 Chemokine receptor Impaired monocyte trafficking (Kuziel et al., 1997) IL-8R Chemokine receptor Impaired neutrophil migration; possibly, expansion of B cells (Cacalano et al., 1995)

ribosylation by pertussis toxin, which leads to their inac- sensitive to pertussis toxin (Huang et al., 1995). The tivation. Sensitivity of a cellular function to pertussis calcium-permeable cation channel CD20, which is highly toxin suggests receptor coupling to one of these G pro- expressed by mature B cells, can be activated by Gi␣2, teins. Some GPCRs activate MAPK through a pertussis suggesting that a Gi␣2-linked GPCR may regulate cal- toxin–sensitive mechanism that is not mediated by Gi␣ cium influx into B cells (Kanzaki et al., 1997). Gi-mediated but rather by associated ␤␥ subunits. In addition, Gi/o signals may also affect the induction of lymphocyte ␤␥ subunits account for the pertussis toxin–dependent apoptosis, since pertussis toxin treatment inhibits acti- activation of PLC (reviewed by Offermanns and Simon, vation-induced apoptosis of monocytes, thymocytes, 1996). PLC catalyzes the hydrolysis of phosphatidyl-4,5- pre-B leukemia cells, T cells, and natural killer cells bisphosphate, generating the second messengers inosi- (Ramirez et al., 1994; Carracedo et al., 1995). Also impli- tol-1,4,5-trisphosphate (IP3) and diacylglycerol. Among cating Gi␣2-coupled receptors in the regulation of T cell -/- the different families of enzymes with PLC activity, the function is the finding that Gi␣2 mice have increased four isoforms of the PLC␤ family are specifically regu- numbers of single-positive thymic T cells with high- lated by heterotrimeric G proteins. Gi␣ subunits also intensity CD3 staining and hyperresponsive T cells in modulate certain potassium and calcium channels. Ex- their periphery (Table 2). Although of unknown signifi- posure of erythroid progenitors to erythropoietin in- cance, Gi␣2 and Gi␣3 have been found associated with the creases cytosolic calcium, apparently through a Gi␣2- glycosylphosphatidylinositol-anchored proteins CD59, activated, voltage-independent calcium channel (Miller CD48, and Thy-1 (Solomon et al., 1996). et al., 1996). All Gq subfamily members can activate all four PLC␤

Gi␣ subunits are present in all lymphoid cell lines and isoforms, but in a pertussis toxin–independent man- primary cells that have been examined (Grant et al., ner and with a rank order different from that of Gi/o ␤␥

1997). Gi␣, predominantly Gi␣2, expression is highest in subunits (reviewed by Fields and Casey, 1997). Al- proliferating lymphoid cell lines, and large in vivo acti- though the molecular mechanisms remain obscure, vated B cells have higher levels than do resting B cells. some GPCRs activate MAPK via a Gq-stimulated pro- No Gi␣1 expression is found in immature B or T cells, tein kinase C–dependent pathway (Hawes et al., 1995). although it is induced during B cell differentiation. Nu- GTPase-deficient forms of Gq are oncogenic in NIH3T3, merous studies implicate Gi-signaling in the regulation of although with low potency. In an illustration of the im- -/- lymphocyte function. Gi-coupled receptors clearly regu- portance of Gq-mediated signaling, platelets from Gq␣ late lymphocyte migration. Treatment of lymphoid cells mice fail to aggregate, generate IP3, or mobilize calcium -/- with pertussis toxin blocks their binding to high endothe- in response to physiological activators. Newborn Gq␣ lial venules and their entrance into lymph nodes and mice often die of intraabdominal bleeding (Offermanns splenic white pulp (Spangrude et al., 1984; Bargatze and et al., 1997a).

Butcher, 1993; Cyster and Goodnow, 1995). A Gi-coupled Gq and G11 ␣ subunits are thought to be expressed receptor may mediate B cell differentiation because a B ubiquitously (Spicher et al., 1994); however, expression cell differentiation factor (446-BCDF)–triggered calcium levels vary markedly in lymphoid cell lines. Of 13 B and flux and enhancement of immunoglobulin secretion are T cell lines tested as well as in thymocytes, tonsil B and Immunity 4

Figure 2. MAP Kinase Activation by Recep- tor Tyrosine Kinases and GPCRs Several mechanisms by which agonists for either tyrosine receptor kinases (RTK, or antigen receptor complex) or GPCRs can activate MAPK. The RGS proteins can negatively reg-

ulate signaling through either Gi-orGq-linked

receptors. Gq can activate both Btk or PLC␤.

Gi␣-associated ␤␥ subunits can active both PI3K␥ and PLC␤. The activation of a Src family kinase can result in agonist independent phosphorylation of an RTK and eventual activation of Ras and the MAPK pathway.

T cells, and splenic B cells, only Jurkat cells and 2 B GTPase Ras, which activates pathways that regulate cell lines expressed Gq␣.G11␣ was found in mature B cellular growth and differentiation (reviewed by Katz and cells and B cell lines and in Jurkat cells, but not in McCormick, 1997). A major consequence of Ras activa- thymocytes or primary T cells (Grant et al., 1997). Impli- tion is stimulation of the MAPK pathway. Growth factor cating Gq␣ in B cell and mast cell function, Gq␣ activates receptors, lymphocyte antigen receptors, and GPCRs, Bruton’s tyrosine kinase (Btk) (Bence et al., 1997). An- which couple to pertussis toxin–sensitive G proteins, other Gq␣ subfamily member, G16␣ (G15␣ in the mouse), utilize Ras for this purpose (Figure 2). Growth factor is expressed in most hematopoietic cells and found at receptors that possess intrinsic tyrosine kinase activity high levels in progenitor B cells. A progressive down- undergo ligand-induced autophosphorylation, creating regulation of G16␣ accompanies B cell differentiation binding sites for docking proteins containing a Src (Amatruda et al., 1991; Mapara et al., 1995). Relatively homology 2 (SH2) domain, leading to the recruitment few GPCRs are known to couple to G16␣, although in of Ras exchange factors and Ras activation. Although the hemapoietic cell line HEL purine receptors do so lymphocyte antigen receptors lack intrinsic tyrosine ki- (Baltensperger and Porzig, 1997) nase activity, they utilize nonreceptor protein tyrosine The downstream targets of the G12 subfamily mem- kinases to assemble a signaling complex for Ras activa- bers are largely undefined, although the expression of tion (reviewed by Berridge, 1997). Similarly, GPCR-medi- GTPase-deficient forms has provided some hints (re- ated Ras activation requires tyrosine kinases. Free Gi viewed by Dhanasekaran and Dermott, 1996; Fields and ␤␥ subunits likely activate Src family kinases, leading Casey, 1997). The activation of the small GTPases Ras to the phosphorylation of adapter proteins and members ϩ ϩ and Rho, the SAPK pathway, Na /H exchangers, and of the focal adhesion kinase family (reviewed by Offer- focal adhesion assemblies have been observed. Re- manns and Simon, 1996; Kranenburg et al., 1997). cently, the induction of nitric oxide synthase has been Several GPCR ligands trigger the tyrosine phosphory- shown (Kitamura et al., 1996). The GTPase-deficient lation of Pyk2, a focal adhesion kinase family member, forms of G and G transcriptionally activate several 12␣ 13␣ and its association with activated Src. Since dominant primary response genes, stimulate mitogenesis, and po- negative forms of Grb2, Sos, and Pyk2 block MAPK tently transform NIH3T3 and Rat-1 fibroblasts (Voyno- activation, Pyk2 and Src activation may link G- and G - Yasenetskaya et al., 1994; Xu et al., 1994). Disruption i q coupled receptors to the MAPK pathway in certain cell of the G gene in mice is lethal, since endothelial cells 13␣ types (Dikic et al., 1996; Della Rocca et al., 1997). Be- in these mice fail to develop into an organized vascular cause T cell receptor (TCR) cross-linking also induces system. In addition, G -/- embryonic fibroblasts migrate 13␣ Pyk2 activation in T cells, Pyk2 potentially serves as a poorly in response to thrombin (Offermanns et al., 1997b). convergence point for TCR and GPCR signaling (Qian et al., 1997). In some cell types, phosphatidylinositol Whereas the G12␣ proteins are thought to be expressed ubiquitously, the screening of a panel of lymphoid cells 3-kinase-␥ (PI3K␥) may be required for G␤␥-dependent MAPK activation (Lopez-Ilasaca, 1997). lines by immunoblotting failed to detect G13␣ (Grant et In B cells, genetic evidence supports the existence al., 1997). However, G13␣ mRNA transcripts have been found in lymphoid cell lines by reverse transcription– of a ligand–GPCR–triggered tyrosine kinase cascade, polymerase chain reaction (Kabouridis et al., 1995). The which precedes MAPK activation (Wan et al., 1997). Based on selective gene targeting in an Avian B lym- receptors that couple to G12 and G13 are largely unidenti- fied, and their roles in chemokine signaling as well as phoma cell line, MAPK activation through Gi-coupled lymphocyte function are unknown. receptors requires Btk and Syk whereas Gq-coupled receptors require Csk, Lyn, and Syk. Finally, Gi-mediated Heterotrimeric G Protein Signaling: signaling may activate MAPK by inducing ligand-inde- Convergence and Cross-Talk pendent tyrosine phosphorylation of a receptor tyrosine Within cells certain key molecules integrate signals from kinase such as the EGF receptor, which then assembles parallel signaling pathways. A good example is the small a signaling complex (Daub et al., 1996; Luttrell et al., Review 5

Figure 3. Btk and Gq␣ Hypothetical scheme for the interaction be-

tween Gq and B cell antigen receptor (BCR) complex signaling and Btk activation. Sig- naling through either the BCR complex or a

Gq-linked GPCR likely activates Btk. The possibility that BCR signaling stimulates Gq␣ tyrosine phosphorylation is speculative. See the review by Kurosaki (1997) for a discussion of the roles of Btk, Lyn, and Syk in B cell function and signaling through the BCR complex. SAPK, stress-activated protein kinase;

PIP2, phosphatidylinositol-4,5-bisphosphate;

DAG, diacylglycerol; IP3, inositol-1,4,5-trisphosphate.

1997). An intriguing possibility is that ligand–GPCR bind- A largely unexplored area is the simultaneous impact ingmay trigger the tyrosine phosphorylation of members of chemokine and antigen receptor signaling. Some evi- of the antigen receptor complexes, thereby modifying dence exists that chemokines and antigen can costimu- antigen receptor signaling. late the activation of human T cells and that endogenous Another important site of signal integration in the cell chemokine production during T cell activation may have is the activation of PLC and the generation of IP3. Inter- a costimulatory role (Taub et al., 1996). The recent recog- estingly, suboptimal stimulation by a Gq-linked receptor nition that the human immunodeficiency virus (HIV) not ligand can markedly augment the rise in intracellular only uses the CXCR4 and CCR5 chemokine receptors calcium in response to a Gi-coupled receptor ligand as coreceptors (reviewed by Broder and Collman, 1997), (Carroll et al., 1995). Thus, preexposure to suboptimal but also probably transduces signals through them, concentrations of a Gq-linked receptor ligand may aug- indicates that investigation of the consequences of ment the biologic response observed following signaling simultaneous and temporally dissociated TCR, CD4, through a chemokine receptor, many of which are Gi and chemokine signaling may provide some insights coupled. Conversely, prolonged Gq-mediated signaling into the pathogenesis of HIV-induced immune dysfunc- down-regulates IP3 receptor levels, desensitizing the cell tion (Weissman et al., 1997). to subsequent signals that activate the IP3/IP3 receptor system (Qian et al., 1994; Honda et al., 1995). There are several examples of cross-talk between G Heterotrimeric G Protein Signaling: Fine-Tuning protein–linked and other signaling pathways in lym- Responses by RGS Proteins phocytes, and innumerable possibilities for interactions. Cells have regulatory mechanisms that limit the duration For example, TCR signaling has been reported to acti- and sensitivity of G protein signaling (reviewed by Bohm et al., 1997). Several molecular mechanisms inhibit het- vate Gq␣ (induce exchange of GTP for GDP) and result in its physical association with CD3⑀ (Stanners et al., erotrimeric G protein signaling. Kinases can phosphory- late GPCRs, allowing members of the arrestin family to 1995). Since Gq␣ and G11␣ apparently require tyrosine phosphorylation for their activation, triggering the lym- bind the phosphorylated receptor, precluding subse- phocyte antigen receptor and tyrosine kinase activation quent G protein activation. This rapidly inactivates signaling. Also, GPCR expression levels decline following could result in Gq␣ and/or G11␣ phosphorylation, thus augmenting GPCR-mediated Gq/11 signaling (Liu et al., exposure to ligand. This likely assists in long-term de- 1996; Umemori et al., 1997). In addition, as mentioned sensitization. Heterotrimeric G protein signaling can above, Gq␣ activates Btk, and G␤␥ subunits have been also be inhibited by proteins that activate the intrinsic reported to bind and activate Btk (Tsukada et al., 1994; GTPase activity of G␣ subunits. GTPase activators, or Langhans-Rajasekaran et al., 1995). Thus, engagement GAPs, were first described for the small GTPase proteins of either the B cell antigen receptor or a Gq-linked GPCR Ras and EF-Tu; however, a newly described family of could activate Btk in B cells (Figure 3). In T cells, Gq- proteins can act as GAPs for G␣ subunits. mediated signaling triggers the translocation of nuclear Genetic studies in yeast, Caenorhabditis elegans, and factor of activated T cells (NF-AT) to the nucleus, provid- Aspergillus nidulans first identified the RGS proteins, inganother mechanism by which TCR- and GPCR-linked which function to quantitatively limit heterotrimeric G signaling pathways may communicate (Boss et al., protein signaling in those organisms (reviewed by Dohl- 1996). Other examples of cross-talk include the inhibi- man and Thorner, 1997; Koelle, 1997). Independently, a tion by Gs-induced increases in cAMP of TCR-triggered mammalian RGS protein termed GAIP was discovered IL-2 promoter activity and IL-6–induced Jak/STAT acti- to interact with G␣ subunits, and four mammalian RGS vation (Ohtsuka et al., 1996; Sengupta et al., 1996). Fi- proteins were shown to substitute functionally for Sst2p, nally, the chemokine RANTES can activate the tyrosine a yeast RGS protein (DeVries et al., 1995; Druey et al., kinase ␨–associated protein-70 (ZAP-70), which is nor- 1996). Based on sequence similarities among the first mally associated with TCR signaling (Bacon et al., 1996). described RGS family members, additional RGS family Immunity 6

members were rapidly identified (DeVries et al., 1995; as GAPs for Gi␣ subfamily members, some RGS proteins Druey et al., 1996; Koelle and Horvitz, 1996) The mam- have clear preferences for particular subfamily mem- malian and C. elegans RGS proteins each contains a bers. In addition, very significant differences in the ability conserved region of approximately 120 amino acids that of RGS proteins to GAP Gq␣ are now apparent (RGS3 Ͼ has been termed the RGS domain, or RGS box. RGS4 ϾϾ GAIP). While further specificity of RGS pro- Biochemical studies have shown that several RGS teins for individual G␣ subunits will undoubtedly be proteins interact directly with Gi␣ and Gq␣; however, no found, the functional specificity of RGS proteins proba- physical interaction with a Gs␣ or G12␣ subfamily member bly also results from their differing patterns of ex- has been reported (Berman et al., 1996a, 1996b; Chen pression and induction and their interactions with other et al., 1996; Dohlman et al., 1996; Hunt et al., 1996; factors. For example, some RGS proteins are broadly Watson et al., 1996; Hepler et al., 1997; Huang et al., distributed, while others are much more restricted (RGS1 1997; Neill et al., 1997). Although RGS proteins do not is predominantly found in B lymphocytes; RGS4 is found affect the time course of nucleotide binding to Gi␣ sub- in neural tissue; and RGS3 is widely distributed) (Hong units, they can potently stimulate the intrinsic rate of Gi␣ et al., 1993; Druey et al., 1996). Detailed expression GTP hydrolysis—in other words, behave as GAPs. To studies for the RGS proteins have not been reported, date, no RGS protein has been shown to act as a GAP although several RGS proteins, including RGS1, RGS2, for a G12␣ or Gs␣. Analysis of the nucleotide dependence RGS3, RGS4, RGS14, and RGS16, are known to be ex- of the binding of RGS4 and RGS1 to Gi␣ revealed that pressed in lymphocytes. RGS proteins bind poorly to GDP- and GTP-bound Some RGS proteins are inducible, since signals gener- forms, but with high affinity to Gi␣ subunits in the GDP ated by GPCR themselves can augment RGS protein Ϫ and AIF4 conformation. This conformation mimics the levels. For example, platelet-activating factor (PAF) trig- transition state for GTP hydrolysis (Coleman et al., 1994). gers RGS1 expression in B cells (Druey et al., 1996). The

Subsequent cocrystallization of RGS4 and Gi␣1 in the constitutive expression of an RGS protein may set a Ϫ GDP and AIF4 conformation revealed that the RGS do- threshold for GPCR signaling, while its induction may main forms a four-helix bundle that directly contacts assist in desensitization. Sst2 serves such a dual role the Gi␣ surface at the three so-called “switch regions.” in yeast. Loss-of-function mutants are hypersensitize to These regions undergo the greatest conformational pheromone, and whereas wild-type yeast desensitize to change during the GTPase cycle and contain residues pheromone, Sst2 mutants do not. Consistent with this critical for GTP hydrolysis. Specific amino acids in RGS4 dual role, Sst2 is expressed at a low constitutive level stabilize the switch region residues in the transition in wild-type yeast and is transcriptionally induced by state, providing the likely mechanism by which RGS pheromone exposure in a time course that parallels the proteins stimulate Gi␣ GTP hydrolysis (Tesmer et al., desensitization (reviewed by Dohlman and Thorner, 1997). A recent mutagenesis study of RGS4 in which 1997). RGS proteins may also be induced by signals the amount of binding of RGS4 mutants to Gi␣1 in the generated through non-GPCR receptors, thereby pro- Ϫ GDP and AIF4 conformation correlated with their GAP viding another mechanism by which a non-GPCR signal- activity is consistent with those findings (Druey and ing pathway might modulate GPCR signaling. Kehrl, 1997). The interaction with other proteins besides G proteins While considerable insight into the biochemistry of may also modulate RGS function. RGS3, RGS7, RGS12, RGS proteins has been achieved, their physiologic roles and RGS14 are substantially larger proteins than the in mammalian cells remain to be determined. Yeast, C. prototypic RGS1 and RGS2 (Druey et al., 1996; Koelle elegans, and Aspergillus nidulans, which lack an RGS and Horvitz, 1996; Snow et al., 1997). Both RGS3 and protein, show clear phenotypes; however, no phenotype RGS12 have regions predicted to assume a coiled coil; related to a mammalian RGS family member has been such domains often mediate interactions with proteins reported (reviewed by Dohlman and Thorner, 1997; of the cellular cytoskeleton. The RGS12 coil-coiled re- Koelle, 1997). Both transient and permanent transfection gion resembles that of rhopilin, a Rho-interacting pro- of RGS family members into mammalian cell lines impairs tein, while RGS14 was isolated as an Rap1/2 interactive ligand–GPCR signaling. RGS4 potently inhibits MAPK acti- protein in a yeast hybrid screen. Thus, the regulation of vation in response to IL-8 stimulation, a Gi-mediated G␣ subunits by RGS12 and RGS14 may be in part under response (Druey et al., 1996). Both RGS4 and RGS3 the control of the small GTPases Rho and Rap1/2, pro- markedly impair signaling through pathways that utilize viding a connection between small GTPases and the

Gq (Hepler et al., 1997; Neill et al., 1997; Yan et al., 1997). heterotrimeric G proteins. RGS4 and probably other RGS proteins may behave as effector antagonists for Gq␣-mediated signals by binding to Gq␣ in its GTP-bound state. A recent report suggested Modulation of Heterotrimeric G Protein that a splice variant of RGS3 inhibits Gs-mediated sig- Signaling by RGS Proteins: Potential nals, although no mechanism was delineated (Chat- Importance in Immune Responses terjee et al., 1997). In summary, there are good reasons How might RGS proteins be important in lymphocyte to think that RGS proteins regulate both the magnitude and inflammatory cell function? Certainly, chemokine and duration of signaling through Gi- and Gq-linked re- receptor signaling is a prime arena for the RGS proteins ceptors; however, whether they regulate GPCRs that to modulate immune and inflammatory cell function. signal through either G12 or Gs requires clarification. Most of the chemokine receptors couple through Gi and

Mammals have at least 18 RGS family members. Al- occasionally Gq (Kuang et al., 1995), both targets of though most of the tested RGS family members can act RGS proteins. The constitutive level of RGS proteins in Review 7

lymphoid and inflammatory cells likely sets a threshold populations of lymphocytes. Such a technique may for the response to chemokines, while their induction allow a direct test of the effects of an RGS protein in a impairs subsequent responses to chemokines. This particular cell system. To explore what role various G␣ type of regulation may help target lymphoid and inflam- subfamily members play in lymphocyte function, trans- matory cells to particular sites and keep them localized genic expression of GTPase-deficient forms of different there, despite the continued exposure to chemokines G␣ subunits may provide useful insights. Finally, as al- and chemoattractants. The importance of chemokines ready witnessed in the results of the initial chemokine and chemokine receptors in B lymphocyte function has and chemokine receptor gene-targeting experiments been underscored by observations made with SDF-1– (Table 2), the analysis of these and other mice in which and BRL1-null mice (Table 2). The chemokine SDF-1, GPCRs or ligands have been targeted should provide which interacts with the CXCR4 receptor, is necessary fertile ground for understanding the physiologic roles of for B lymphocyte development. SDF-1 mutant mice have these receptors and their signaling pathways. a major defect in both fetal liver and bone marrow B lymphopoiesis (Bleul et al., 1996; Nagasawa et al., 1996; Conclusions D’Apuzzo et al., 1997). BRL1 is a heptahelical transmem- The RGS proteins provide a mechanism by which cells brane receptor and a putative chemokine receptor: it can regulate both the duration and the magnitude of a contains a characteristic sequence motif at the end of signal generated through a heterotrimeric G protein. transmembrane domain 3 (Dobner et al., 1992). BRL1 Such fine-tuning is undoubtedly essential for the finely mutant mice lack inguinal lymph nodes, phenotypically orchestrated events that occur in response to the che- normal Peyer’s patches, and germinal centers (Forster mokines, hormones, and neuropeptides, which signal et al., 1996). By modulating signalingthrough the CXCR4 through GPCRs. Furthermore, certain key intracellular and BRL1 receptors, RGS proteins should influence the molecules such as Ras integrate output signals gener- development and differentiation of B lymphocytes. ated through heptahelical, antigen, and cytokine recep- Besides the chemokine receptors, signaling through tors. Discovering these key molecules and understand- chemoattractant receptors such as the receptors for ing how they discriminate between relevant signals and PAF, formyl-methionyl-leucyl-phenylalanine (FMLP), C5a, irrelevant background noise to coordinate the cellular lysophosphatidic acid (LPA), and leukotriene B4 can responses that eventually lead to humoral and cellular potently affect lymphocyte and inflammatory cells (Camp- immunity remains a sizable challenge for the future. bell et al., 1996). For example, PAF has dramatic effects on B lymphocytes. It induces the transcription of imme- diate early genes; activates NF-␬B (nuclear factor ␬B); References triggers tyrosine phosphorylation of Fyn, Lyn, PI3K, and PLC␥1; triggers cytokine secretion; activates MAPK; Amatruda, T.T., Steele, D.A., Slepak, V.Z., and Simon, M.I. (1991). G␣16, a G protein ␣ subunit specifically expressed in hematopoietic and increases immunoglobulin secretion (Mazer et al., cells. Proc. Natl. Acad. Sci. USA 88, 5587–5591. 1991; Kuruvilla et al., 1994; Smith and Shearer, 1994; Apasov, S.G., Koshiba, M., Chused,T.M., and Sitkovsky, M.V. (1997). Franklin et al., 1995; Kravchenko et al., 1995). Demon- Effects of extracellular ATP and adenosine on different thymocyte strating the potential for RGS proteins to regulate PAF subsets. J. Immunol. 158, 5095–5105. receptor signaling, the constitutive expression of RGS1 Bacon, K.B., Szabo, M.C., Yssel, H., Bolen, J.B., and Schall, T.J. in a B lymphocyte cell line impaired PAF-induced MAPK (1996). RANTES induces tyrosine kinase activity of stably complexed activation (Druey et al., 1996). Another potential RGS p125FAK and ZAP-70 in human T cells. J. Exp. Med. 184, 873–882. target in lymphocytes is LPA-mediated signaling. LPA Baixeras, E., Garcia-Lozano, E., and Martinez, C. (1996). Decrease activates Jurkat T cells to proliferate in serum-free me- in cAMP levels promoted by CD48-CD2 interaction correlates with dia, induces a calcium flux, and augments IL-2 produc- inhibition of apoptosis in B cells. Scand. J. Immunol. 43, 406–412. tion (Xu et al., 1995). There is also increasing evidence Baltensperger, K., and Porzig, H. (1997). The P2U purinoceptor that neuroendocrine inputs influence immune and in- obligatorily engages the heterotrimeric G protein G16 to mobilize intracellular Ca2ϩ in human erythroleukemia cells. J. Biol. Chem. flammatory responses (reviewed by Goetzl et al., 1995). 272, 10151–10159. For example, signaling through purinergic receptors af- Bargatze, R.F., and Butcher, E.C. (1993). Rapid G protein-regulated fects different thymocyte subsets, and substance P acti- activated event involved in lymphocyte binding to high endothelial vates T cells and monocytes and induces Bcell immuno- venules. J. Exp. Med. 178, 367–372. globulin synthesis (Kavelaars et al., 1994; Apasov et al., Bence, K., Ma, W., Kozasa, T., and Huang, X.-Y. (1997). Direct stimu- 1997). Since these molecules signal via GPCRs, the RGS lation of Bruton’s tyrosine kinase by Gq-protein ␣-subunit. Nature proteins should modulate signaling through these re- 389, 296–299. ceptors as well. Berman, D.M., Wilkie, T.M., and Gilman, A.G. (1996a). GAIP and Several types of experiments will be useful for examin- RGS4 are GTPaseϪactivating proteins (GAPs) for the Gi subfamily ing the roles of heterotrimeric G protein signaling and of G protein ␣ subunits. Cell 86, 445–452. RGS proteins in lymphocyte function in vivo. One ap- Berman, D.M., Kozasa, T., and Gilman, A.G. (1996b). The GTPase- proach will be targeted deletion of the RGS family mem- activating protein RGS4 stabilizes the transition state for nucleotide hydrolysis. J. Biol. Chem. 271, 27209–27212. bers expressed in lymphocytes. A caveat of this method Berridge, M.J. (1997). Lymphocyte activation in health and disease. is that the phenotypes may be subtle, particularly in light Crit. Rev. Immunol. 17, 155–178. of the large number of RGS proteins, which may be able Bleul, C.C., Farzan, M., Choe, H., Parolin, C., Clark-Lewis, I., So- to compensate for the lack of a single RGS protein. In droski, J., and Springer, T.A. (1996). The lymphocyte chemoattrac- another approach, various RGS proteins may be trans- tant SDF-1 is a ligand for LESTR/fusion and blocks HIV-1 entry. genically overexpressed or misexpressed in different Nature 382, 829–833. Immunity 8

Bohm, S.K., Grady, E.F., and Bunnett, N.W. (1997). Regulatory mech- Dohlman, H.G., and Thorner, J. (1997). RGS proteins and signaling anisms that modulate signaling by G-protein-coupled receptors. by heterotrimeric G proteins. J. Biol. Chem. 272, 3871–3874. Biochem. J. 322, 1–18. Dohlman, H.G., Song, J., Ma, D., Courchesne, W.E., and Thorner, Boss, V., Talpade, D.J., and Murphy, T.J. (1996). Induction of NFAT- J. (1996). Sst2, a negative regulator of pheromone signaling in the mediated transcription by Gq-coupled receptors in lymphoid and yeast Saccharomyces cerevisiae: expression, localization, and ge- non-lymhoid cells. J. Biol. Chem. 271, 10429–10432. netic interaction and physical association with Gpa1 (the G-protein Broder, C.C., and Collman, R.G. (1997). Chemokine receptors and ␣ subunit). Mol. Cell. Biol. 16, 5194–5209. HIV. J. Leukoc. Biol. 62, 20–29. Druey, K.M., and Kehrl, J.H. (1997). Inhibition of RGS function by Cacalano, G., Lee, J., Kikly, K., Ryan, A.M., Pitts-Meek, S., Hultgren, two mutant RGS4 proteins, Proc. Natl. Acad. Sci. USA, in press. B., Wood, W.I., and Moore, M.W. (1995). Neutrophil and B cell expan- Druey, K.M., Blumer, K.J., Kang, V.H., and Kehrl, J.H. (1996). Inhibi- sion in mice that lack the murine IL-8 receptor homolog. Science tion of G-protein-mediated MAP kinase activation by a new mamma- 269, 1590–1591. lian gene family. Nature 379, 742–746. Campbell, J.J., Qin, S., Bacon, K.B., Mackay, C.R., and Butcher, Fedyk, E.R., and Phipps, R.P. (1996). Prostaglandin E2 receptors of E.C. (1996). Biology of chemokine and classical chemoattractant the EP2 and EP4 subtypes regulate activation and differentiation of receptors: differential requirements for adhesion-triggering versus mouse B lymphocytes to IgE-secreting cells. Proc. Natl. Acad. Sci. chemotactic responses in lymphoid cells. J. Cell Biol. 134, 255–266. USA 93, 10978–10983. Carracedo, J., Ramirez, R., Marchetti, P., Pintado, O.C., Baixeras, Fedyk, E.R., Adawi, A., Looney, R.J., and Phipps, R.P. (1996). Regu- E., Martinez, C., and Kroemer, G. (1995). Pertussis toxin-sensitive lation of IgE and cytokine production by cAMP: implications for GTP-binding proteins regulate activation-induced apoptotic cell extrinsic asthma. Clin. Immunol. Immunopathol. 81, 101–113. death of human natural killer cells. Eur. J. Immunol. 25, 3094–3099. Fields, T.A., and Casey, P.J. (1997). Signaling functions and bio- Carroll, R.C., Morielli, A.D., and Peralta, E.G. (1995). Coincidence chemical properties of pertussis toxin-resistant G-proteins. Bio- detection at the level of phospholipase C activation mediated by chem. J. 321, 561–571. the m4 muscarinic acetylcholine receptor. Curr. Biol. 5, 536–544. Forster, R., Mattis, A.E., Kremmer, E., Wolf, E., Brem, G., and Lipp, Chatterjee, T.K., Eapen, A.K., and Fisher, R.A. (1997). A truncated M. (1996). A putative chemokine receptor, BLR1, directs B cell mi- form of RGS3 negatively regulates G protein-coupled receptor stim- gration of defined lymphoid organs and specific anatomic compart- ulation of adenylyl cyclase and phosphoinositide phospholipase C. ments of the spleen. Cell 87, 1037–1047. J. Biol. Chem. 272, 15481–15487. Franklin, R.A., Tordai, A., Mazer, B., Terada, N., Lucas, J., and Gel- Chen, C.-K., Wieland, T., and Simon, M.I. (1996). RGS-r, a retinal fand, E.W. (1995). Platelet activating factor activates MAPK and specific RGS protein, binds an intermediate conformation of trans- increases in intracellular calcium via independent pathways in B ducin and enhances recycling.Proc. Natl. Acad. Sci. USA 93, 12885– lymphocytes. Biochem. Biophys. Res. Comm. 209, 1111–1118. 12889. Gao, J.-L., Wynn, T.A., Chang, Y, Lee, E.J., Broxmeyer, H.E., Cooper, S., Tiffany, H.L. Westphal, H., Kwon-Chung, J., and Murphy, P.M. Chen, J., and Iyengar, R. (1994). Suppression of Ras-induced transformation of NIH 3T3 cells by activated G␣s. Science 263, 1278–1281. (1997). Impaired host defense, hematopoiesis, granulomatous in- flammation and type 1-type 2 cytokine balance in mice lacking CC Coleman, D.E., Berghuis, A.M., Lee, E., Linder, M.E., Gilman, A.G., chemokine receptor 1. J. Exp. Med. 185, 1959–1968. and Sprang, S.R. (1994). Structures of active conformations of Gi␣1 Gilman, A.G. (1995). G proteins and regulation of adenylyl cyclase. and the mechanism of GTP hydrolysis. Science 265, 1405–1412. Biosci. Rep. 15, 65–97. Cook, D.N., Beck, M.A., Coffman, T.M., Kirby, S.L., Sheridan, J.F., Goetzl, E.J., Xia, M., Ingram, D.A., Kishiyama, J.L., Kaltreider, H.B., Pragnell, I.B., Smithies, O. (1995). Requirement of MIP-1 alpha for Byrd, P.K., Ichikawa, S., and Sreedharan, S.P. (1995). Neuropeptide an inflammatory response to viral infection. Science 269, 1583–1585. signaling of lymphocytes in immunological responses. Int. Arch. Cyster, J.G., and Goodnow, C.C. (1995). Pertussis toxin inhibits Allergy Immunol. 107, 202–204. migration of B and T lymphocytes into splenic white pulp cords. J. Grant, K.R., Harnett, W., Millignan, G., and Harnett, M.M. (1997). Exp. Med. 182, 581–586. Differential G-protein expression during B- and T-cell development. Daeron, M. (1997). Fc receptor biology. Annu. Rev. Immunol. 15, Immunology 90, 564–571. 203–234. Hamm, H.E., and Gilchrist, A. (1996). Heterotrimeric G proteins. Curr. D’Apuzzo, M., Rolink, A., Leotscher, M., Hoxie, J.A., Clark-Lewis, I., Opin. Cell Biol. 8, 189–196. Melchers, F., Baggiolini, M., and Moser, B. (1997). The chemokine Hawes, B.E., van Biesen, T., Koch, W.J., Luttrell, L.M., and Lefkowitz, SDF-1, stromal cell-derived factor 1, attracts early stage B cell pre- R.J. (1995). Distinct pathways of G - and G -mediated mitogen-acti- cursors via the chemokine receptor CXCR4. Eur. J. Immunol. 27, i q vated protein kinase activation. J. Biol. Chem. 270, 17148–17153. 1788–1793. Hepler, J.R., Berman, D.M., Gilman, A.G., and Kozasa, T. (1997). Daub, H., Weiss, F.U., Wallasch, C., and Ullrich, A. (1996). Role of RGS4 and GAIP are GTPase-activating proteins for G and block transactivation of the EGF receptor in signaling by G-protein-cou- q␣ activation of phospholipase C␤ by ␥-thio-GTP-G . Proc. Natl. Acad. pled receptors. Nature 379, 557–560. q␣ Sci. USA 94, 428–432. Della Rocca, G.J., van Biesen, T., Daaka, Y., Luttrell, D.K., Luttrell, Honda, Z., Takano, T., Hirose, N., Suzuki, T., Muto, A., Kume, S., L.M., and Lefkowitz, R.J. (1997). Ras-dependent mitogen-activated Mikoshiba, K., Itoh, K., and Shimizu, T. (1995). Gq pathway desensi- protein kinase activation by G protein-coupled receptors. Conver- tizes chemotactic receptor-induced calcium signaling via inositol gence of G- and G -mediated pathways on calcium/calmodulin, i q trisphosphate receptor down-regulation. J. Biol. Chem. 270, 4840– Pyk2, and Src kinase. J. Biol. Chem. 272, 19125–19132. 4844. DeVries, L., Mousli, M., Wurmser, A., and Farquhar, M.G. (1995). Hong, J.X., Wilson, G.L., Fox, C.H., and Kehrl, J.H. (1993). Use of GAIP, a protein that specifically interacts with the trimeric G protein subtractive cloning to isolate B lymphocyte specific genes: charac- G␣i3, is a member of a protein family with a highly conserved core terization of a novel B cell activation gene. J. Immunol. 150, 3895– domain. Proc. Natl. Acad. Sci. USA 92, 11916–11920. 3904. Dhanasekaran, N., and Dermott, J.M. (1996). Signaling by the G12 Hornquist, C.E., Lu, X., Rogers-Fani, P.M., Rudolph, U., Shappell, class of G proteins. Cell Signal. 8, 235–245. S., Birnbaumer, L., and Harriman, G.R. (1997). G␣i2-deficient mice Dikic, I., Tokiwa, G., Lev, S., Courtneidge, S.A., and Schlessinger, J. with colitis exhibit a local increase in memory CD4ϩ T cells and (1996). A role for Pyk2 and Src in linking G-protein-coupled receptors proinflammatory Th1-type cytokines. J. Immunol. 158, 1068–1077. with MAP kinase activation. Nature 383, 547–550. Huang, R., Cioffi, J., Berg, K., London, R., Cidon, M., Maayani, S., Dobner, T., Wolf, I., Emrich, T., and Lipp, M. (1992). Differentiation- and Mayer, L. (1995). B cell differentiation factor-induced B cell specific expression of a novel G protein-coupled receptor from Bur- maturation: regulation via reduction in cAMP. Cell. Immunol. 162, kitt’s lymphoma. Eur. J. Immunol. 22, 2795–2799. 49–55. Review 9

Huang, C., Hepler, J.R., Gilman, A.G., and Mumby, S.M. (1997). Cheung, J.Y. (1996). G-protein alpha subunit Gi(alpha)2 mediates

Attenuation of Gi- and Gq-mediated signaling by expression of RGS4 erythropoietin signal transduction in human erythroid precursors. J. or GAIP in mammalian cells. Proc. Natl. Acad. Sci. USA 94, 6159– Clin. Invest. 98, 1728–1736. 6163. Murphy, P.M. (1996). Chemokine receptors: structure, function and Hunt, T.W., Fields, T.A., Casey, P.J., and Peralta, E.G. (1996). RGS10 role in microbial pathogenesis. Cytokine Growth Factor Rev. 7, is a selective activator of Gia GTPase activity. Nature 383, 175–177. 47–64. Kabouridis, P.S., Waters, S.T., Escobar, S., Stanner, J., and Tsoukas, Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa, C.D. (1995). Expression of GTP-binding protein ␣ subunits in human S., Kitamura, Y., Yoshida, N., Kikutani, H., and Kishimoto, T. (1996). thymocytes. Mol. Cell. Biochem. 144, 45–51. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in Kanzaki, M., Lindorfer, M.A., Garrison, J.C., and Kojima, I. (1997). mice lacking the CXC chemokine PBSF/SDF-1. Nature 382, 635–638. Activation of the calcium-permeable cation channel CD20 by alpha Neer, E.J. (1995). Heterotrimeric G proteins:organizers of transmem- subunits of the Gi protein. J. Biol. Chem. 272, 14733–14739. brane signals. Cell 80, 249–257. Katz, M.E., and McCormick, F. (1997). Signal transduction from mul- Neill, J.D., Duck, L.W., Sellers, J.C., Musgrove, L.C., Scheschonka, tiple Ras effectors. Curr. Opin. Genet. Dev. 7, 75–79. A., Druey, K.M., and Kehrl, J.H. (1997). Potential role for a regulator Kavelaars, A., Jeurissen, F., and Heignen, C.J. (1994). Substance P of G protein signaling (RGS3) in gonadotropin-releasing hormone receptors and signal transduction in leukocytes. Immunomethods (GnRH) stimulated desensitization. Endocrinology 138, 843–846. 5, 41–48. Offermanns, S., and Simon, M.I. (1996). Organization of transmem- Kitamura, K., Singer, W.D., Star, R.A., and Muallem, S. (1996). Induc- brane signaling by heterotrimeric G proteins. Cancer Surv. 27, tion of inducible nitric-oxide synthase by the heterotrimeric G protein 177–198.

G␣13. J. Biol. Chem. 271, 7412–7415. Offermanns, S., Toombs, C.F., Hu, Y.-H., and Simon, M.I. (1997a). Koelle, M.R. (1997). A new family of G-protein regulators - the RGS Defective platelet activation in G␣q-deficient mice. Nature 389, proteins. Curr. Opin. Cell Biol. 9, 143–147. 183–186. Koelle, M.R., and Horvitz, H.R. (1996). EGL-10 regulates G protein Offermanns, S., Mancino, V., Rev.el, J.-P., and Simon, M.I. (1997b). signaling in the C. elegans nervous system and shares a conserved Vascular system defects and impaired cell chemokinesis as a result domain with many mammalian proteins. Cell 84, 115–125. of G␣13 deficiency. Science 275, 533–536. Kranenburg, O., Verlaan, I., Hordijk, P.L., and Moolenaar, W.H. Ohtsuka, T., Kaziro, Y., and Satoh, T. (1996). Analysis of the T-cell (1997). Gi-mediated activation of the Ras/MAP kinase pathway in- activation signaling pathway mediated by tyrosine kinases, protein volves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, kinase C, and Ras protein, which is modulated by intracellular cyclic but not Src nor Shc. EMBO J. 16, 3097–3105. AMP. Biochem. Biophys. Acta 1310, 223–232. Kravchenko, V.V., Pan, Z., Han, J., Herbert, J.-M., Ulevitch, R.J., and O’Shea, J.J. (1997). Jaks, STATs, cytokine signal transduction, and Ye, R.D. (1995). Platelet-activating factor indiuces NF-␬B activation immunoregulation: are we there yet? Immunity 7, 1–11. through a G-protein-coupled pathway. J. Biol. Chem. 270, 14928– Paliogianni, F., Kincaid, R.L., and Boumpas, D.T. (1993). Prostaglan- 14934. din E2 and other cyclic AMP elevating agents inhibit interleukin 2 Kuang, Y., Wu, Y., Jiang, H., and Wu, D. (1995). Selective G protein gene transcription by counteracting calcineurin-dependent path- coupling by C-C chemokine receptors. J. Biol. Chem. 271, 3975– ways. J. Exp. Med. 178, 1813–1817. 3978. Qian, D., and Weiss, A. (1997). T cell antigen receptor signal trans- Kurosaki, T. (1997). Molecular mechanisms in B cell antigen receptor duction. Curr. Opin. Cell. Biol. 9, 205–212. signaling. Curr. Opin. Immunol. 9, 309–318. Qian, D., Lev, S., vanOers, N.S. C., Dikic, I., Schlessinger, J., and Kuruvilla, A., Pielop, C., and Shearer, E.T. (1994). Platelet-activating Weiss, A. (1997). Tyrosine phosphorylation of Pyk2 is selectively factor induces the tyrosine phosphorylation and activation of phos- regulated by fyn during TCR signaling. J. Exp. Med. 185, 1253–1259. pholipase C-␥1, fyn and lyn kinases, and phosphatidylinositol Qian, N.X., Russell, M., Buhl, A.M.,and Johnson, G.L. (1994). Expres- 3-kinase in a human B cell line. J. Immunol. 153, 5433–5442. sion of GTPase-deficient G␣16 inhibits Swiss 3T3 cell growth J. Biol. Kuziel, W.A., Morgan, S.J., Dawson, T.C., Griffin, S., Smithies, O., Chem. 269, 17417–17423. Ley, K., and Maeda, N. (1997). Severe reduction in leukocyte adhe- Ramirez, R., Carracedo, J., Zamzami, N., Castedo, M., and Kroemer, sion and monocyte extravasation in mice deficient in CC chemokine G. (1994). Pertussis toxin inhibits activation-induced cell death of receptor 2. Proc. Natl. Acad. Sci. USA 94, 12053–12058. human thymocytes, pre-B leukemia cells and monocytes. J. Exp. Langhans-Rajasekaran, S.A., Wan, Y., and Huang, X.-Y. (1995). Acti- Med. 180, 1147–1152. vation of Tsk and Btk tyrosine kinases by G protein ␤␥ subunits. Roper, R.L., Ludlow, J.W., and Phipps, R.P. (1994). Prostaglandin E2 Proc. Natl. Acad. Sci. USA 92, 8601–8605. inhibits B lymphocyte activation by a cAMP-dependent mechanism: Liu, W.W., Mattingly, R.R., and Garrison, J.C. (1996). Transformation PGE-inducible regulatory proteins. Cell. Immunol. 154, 296–308. of Rat-1 fibroblasts with the v-src oncogene increases the tyrosine Rothenberg, M.E., MacLean, J.A., Pearlman, E., Luster, A.D., and phosphorylation state and activity of the ␣ subunit of Gq/G11. Proc. Leder, P. (1997). Targeted disruption of the chemokine eotaxin par- Natl. Acad. Sci. USA 93, 8258–8263. tially reduces antigen-induced tissue eosinophilia. J. Exp. Med. 185, Lopez-Ilasaca, M., Crespo, P., Pellici, P.G., Gutkind, J.S., and Wetz- 785–790. ker, R. (1997). Linkage of G protein-coupled receptors to the MAPK Sanders, V.M. (1995). The role of adrenoceptor-mediated signals signaling pathway through PI 3-kinase ␥. Science 275, 394. in the modulation of lymphocyte function. Adv. Neuroimmunol. 5, Luttrell, L.M., Della Rocca, G.J., vanBiesen, T., Luttrell, D.K., and 283–298.

Lefkowitz, R.J. (1997). G␤␥ subunits mediate Src-dpendent phos- Scherer, L.J., Diamond, R.A., and Rothenberg, E.V. (1995). Develop- phorylation of the epidermal growth factor receptor. J. Biol. Chem. mental regulation of cAMP signaling pathways in thymocyte devel- 272, 4637–4644. opment. Thymus 23, 231–257. Mapara, M.Y., Bommert, K., Bargou, R.C., Leng, C., Beck, C., Lud- Sengupta, T.K., Schmitt, E.M., and Ivashkiv, L.B. (1996). Inhibition of wig, W. D., Gierschik, P., and Dorken, B. (1995). G protein subunit cytokines and JAK-STAT activation by distinct signaling pathways.

G␣16 expression is restricted to progenitor B cells during human Proc. Natl. Acad. Sci. USA 93, 9499–9504. B-cell differentiation. Blood 85, 1836–1842. Smith, C.S., and Shearer, W.T. (1994). Activation of NF-␬Band immu- Mazer, B., Domenico, J., Sawami, H., and Gelfand, E.W. (1991). noglobulin expression in response to platelet-activating factor in a Platelet-activating factor induces an increase in intracellular calcium human B cell line. Cell. Immunol. 155, 292–303. and expression of regulatory genes in human B lymphoblastoid Snow, B.E., Antonio, L., Suggs, S., Gutstein, H.B., and Siderovski, cells. J. Immunol. 146, 1914–1920. D.P. (1997). Molecular cloning and expression analysis of rat RGS12 Miller, B.A., Bell, L., Hansen, C.A., Robishaw, J.D., Linder, M.E., and and RGS14. Biochem. Biophys. Res. Comm. 233, 770–777. Immunity 10

Solomon, K.R., Rudd, C.E., and Finberg, R.W. (1996). The association between glycosylphosphatidylinositol-anchored proteins and heterotrimeric G protein ␣ subunits in lymphocytes. Proc. Natl. Acad. Sci. USA 93, 6053–6058. Spangrude, G.J., Braaten, B.A., and Daynes, R.A. (1984). Molecular mechanisms of lymphocyte extravasation. I. Studies of two selective inhibitors of lymphocyte recirculation. J. Immunol. 132, 354–362. Spicher, K., Kalkbrenner, F., Zobel, A., Harhammer, R., Nurnberg,

B., Soling, A., and Schultz, G. (1994). G12 and G13 ␣-subunits are immunochemically detectable in most membranes of various mammalian cells and tissues. Biochem. Biophys. Res. Commun. 198, 906–914. Spiegel, A.M. (1997). The molecular basis of disorders caused by defects in G proteins. Hormone Res. 47, 89–96. Stanners, J., Kabouridis, P.S., McGuire, K.L., and Tsoukas, C.D. (1995). Interaction between G proteins and tyrosine kinases upon T cell receptor CD3-mediates signaling. J. Biol. Chem. 270, 30635– 30642. Stephens, L.R., Eguinoa, A., Erdjument-Bromage, H., Lui, M., Cooke, F., Coadwell, J., Smrcka, A.S., Thelen, M., Cadwallader, K., Tempst, P., et al. (1997). The G beta gamma sensitivity of a PI3K is dependent upon a tightly associated adaptor, p101. Cell 89, 105–114. Tamir, A., Granot, Y., and Isakov, N. (1996). Inhibition of Tlymphocyte activation by cAMP is associated with down-regulation of two parallel mitogen-activated protein kinase pathways, the extracellular signal-related kinase and c-Jun N-terminal kinase. J. Immunol. 157, 1514–1522. Taub, D.D., Turcovski-Corrales, S.M., Key, M.L., Longo, D.L., and Murphy, W.J. (1996). Chemokines and T lymphocyte activation: I. Beta chemokines co-stimulate human T lymphocyte activation in vitro. J. Immunol. 156, 2095–2103. Tesmer, J.J. G., Berman, D.M., Gilman, A.G., and Sprang, S. (1997). Structure of RGS4 bound to AIF4-activated Gi␣1: stabilization of the transition state for GTP hydrolysis. Cell 89, 251–261. Tsukada, S., Simon, M.I., Witte, O.N., and Katz, A. (1994). Binding of ␤␥ subunits of heterorimeric G proteins to the PH domain of Bruton tyrosine kinase. Proc. Natl. Acad. Sci. USA 91, 11256–11260. Umemori, H., Inoue, T., Kume, S., Sekiyama, N., Nagao, M., Itoh, H., Nakanishi, S., Mikoshiba, K., and Yamamoto, T. (1997). Activation of the G protein Gq/11 through tyrosine phosphorylation of the ␣ subunit. Science 276, 1878–1881. Voyno-Yasenetskaya, T.A., Pace, A.M., and Bourne, H.R. (1994).

Mutant ␣ subunits of G12 and G13 proteins induce neoplastic transformation of Rat-1 fibroblasts. Oncogene 9, 2559–2565. Wan, Y., Bence, K., Hata, A., Kurosaki, T., Velillete, A., and Huang, X.Y. (1997). Genetic evidence for a tyrosine kinase cascade preced- ing the mitogen-activated protein kinase cascade in vertebrate G protein signaling. J. Biol. Chem. 4, 17209–17215. Watson, N., Linder, M.E., Druey, K.M., Kehrl, J.H., and Blumer, K.J. (1996). RGS family members: GTPase-activating proteins for heterotrimeric G-protein ␣-subunits. Nature 383, 172–175. Weissman, D., Rabin, R.L., Arthos, J., Rubbert, A., Dybul, M., Ven- katesan, S., Farber, J.M., and Fauci, A.S. (1997). Macrophage-tropic HIV and SIV envelope proteins induce a signal through the CCR5 chemokine receptor. Nature 389, 981–985. Xu, N., Voyno-Yasenetskaya, T., and Gutkind, J.S. (1994). Potent transforming activity of the G13 ␣ subunit defines a novel family of oncogenes. Biochem. Biophys. Res. Commun. 201, 603–609. Xu, Y., Casey, G., and Mills, G.B. (1995). Effect of lysophospholipids on signaling in the human Jurkat T cell line. J. Cell. Physiol. 163, 441–450.

Yan, Y., Chi, P.P., and Bourne, H.R. (1997). RGS4 inhibits Gq-mediated activation of mitogen-activated protein kinase and phosphoinositide synthesis. J. Biol. Chem. 272, 11924–11927.