Eur. J. Biochem. 195,289-303 (1991) 0 FEHS 1991 0014295691000354

Review Sensory transduction in eukaryotes A comparison between Dictyosteliurn and vertebrate cells Peter J. M. Van HAASTERTI, Pim M. W. JANSSENS’ and Christophe ERNEUX3 Department of Biochemistry, University of Groningen, The Netherlands Central Clinical Chemical Laboratory, St. Radboud University Hospital, Nijmegen, The Netherlands Institute of Interdiciplinary Research, School of Medicine, Free University of Brussels, Belgium (Received July 10, 1990) - EJB 90 0822

The organization of multicellular organisms depends on cell -cell communication. The signal molecules are often soluble components in the extracellular fluid, but also include odors and light. A large array of surface receptors is involved in the detection of these signals. Signals are then transduced across the plasma membrane so that enzymes at the inner face of the membrane are activated, producing second messengers, which by a complex network of interactions activate target proteins or genes [ 11. Vertebrate cells have been used to study hormone and neurotransmitter action, vision, the regulation of cell growth and differentiation. Sensory transduction in lower eukaryotes is predominantly used for other functions, notably cell attraction for mating and food seeking. By comparing sensory transduction in lower and higher eukaryotes general principles may be recognized that are found in all organisms and deviations that are present in specialised systems. This may also help to understand the differences between cell types within one organism and the importance of a particular pathway that may or may not be general. In a practical sense, microorganisms have the advantage of their easy genetic manipulation, which is especially advantageous for the identification of the function of large families of signal transducing components.

In this review, we describe sensory transduction in eukaryotic cells, by comparing the microorganism Dictyostelium discoideum (Fig. 1) with vertebrate cells. Signal transduction in these organisms is compared with that in other lower eukaryotes.

amoeba fruiting

Sensory transduction systems, an overview

Cells recognize extracellular signals by several mechanisms: signal molecules may bind to surface receptors transmitting the signal into the cell, signal molecules may diffuse into the cell where they bind to specific receptors, or signal molecules may modulate the transduction of other signals by interfering with specific sensory transduction components. Correspondence to P. J. M. Van Haastert, Department of Biochemistry, University of Groningen, Nijenborgh 16, NL-9747 AG Groningen, The Netherlands Abbreviations. G-protein, GTP-binding regulatory protein; G,, G-protein stimulating adenylate cyclase; Gi, G-protein inhibiting adenylate cyclase; InsP, InsP2, InsP3 (etc.) myo-inositol mono-, bis-, tris- (etc.) phosphates with isomeric numbering (all D-) as appropriate; PtdInsP2, phosphatidylinositol bisphosphate; GTP[yS], 5’-[y-thio]triphosphate; GAP, GTPase-activating factor. Note. After preparation of this review two manuscripts were published demonstrating the presence in Dictyosteliurn of proteintyrosine-kinase genes (J. L. Tan & J. A. Spudich (1990) MoZ. Cell. Biol. 10, 3578 - 3583) and phosphotyrosine-containing proteins (A. Schweiger, 0. Mihalache, A. Muhr & I. Adrian (1990) FEBS Lett. 268,199 - 202).

T

\

UNICELLULAR GROWTH

MULTICELLULAR DIFFERENTIATION

6

pseudoplasmodium

Fig. 1 . Development of D. discoideum. During the life cycle of D.discoideum, the vegetative amoebae divide. Starvation induces cell aggregation. In the multicellular structure, cell differentiation takes place, resulting in the formation of a fruiting body

Sensory transduction by G-protein coupled receptors [2 -41

The signal molecule binds to a surface receptor that has seven putative transmembrane-spanning domains. This recep-

290 tor interacts with one or more G-proteins that are localized Sensory transduction by receptors at the inner face of the plasma membrane. G-proteins are with guanylate cyclase activity composed of three subunits, ap).The /? and y subunits probThe recent cloning of some genes encoding guanylate ably form a permanent complex with each other and are cyclase suggests that this transduction pathway may have required for the modulation of the activity of the CI subunit. The [x subunit is generally thought to be the active species that unique characteristics [21 - 251. The membrane-bound modulates the activity of intracellular effector enzymes. The guanylate cyclase in sea urchin spermatozoa is stimulated by ligand-occupied surface receptor converts the inactive aGDp& peptides secreted by sea urchin eggs, inducing chemoattraction of spermatozoa. A strong species-specificity of secreted complex to the active configuration aGTp By. The following functions have been ascribed to G-proteins : peptides and receptor binding precludes inter-species actistimulation and inhibition of adenylate cyclase, stimulation vation. In mammalian cells, a membrane-bound guanylate of cGMP phosphodiesterase in retina, stimulation of cyclase is activated by atrial natriuretic peptide. In both organphospholipase C, stimulation of phospholipase Az, and regu- isms, membranous guanylate cyclase activity and specific lation of opening or closing of ion channels. The enzymes alter binding of the ligand appears to be present in one protein. the concentration of intracellular second messengers, such The proteins also contains a domain that is highly homologous as CAMP, Ins(1 ,4,5)P3. diacylglycerol, Ca2+. These second to serine and/or tyrosine kinases, but kinase activity has not messengers transduce the signal to target proteins, which are been demonstrated. This putative kinase domain may be involved in the regulation of the catalytic domain because deoften protein kinases. This general scheme of sensory transduction by G-protein- letion of the kinase domain leads to the constitutive activation coupled receptors is found in nearly all eukaryotic organisms, of guanylate cyclase [26]. In D. discoideum guanylate cyclase is activated by extraand many of the proteins have been identified; details will be cellular cAMP [27,28]. The cyclic nucleotide specificity of the discussed below. activation of guanylate cyclase is identical to the specificity of cAMP binding to the surface receptor [29]. This surface Sensory transduction by receptors with tyrosine kinase activity receptor, however, has the structure of a G-protein-coupled In contrast to G-protein-coupled receptors, the receptors receptor with seven putative transmembrane-spanning doof this class have enzymatic activity. The receptor transverses mains and a guanylate cyclase catalytic domain is virtually the membrane only once; the signal molecule binds to the absent [30]. The membrane-bound guanylate cyclase is extracellular domain and, by a mechanism that may involve strongly inhibited by submicromolar CaZf concentrations receptor dimerization, a tyrosine kinase activity in the cyto- [31], which is also observed for mammalian guanylate cyclase plasmic domain of the receptor is stimulated [5-71. This of rod-outer segments [32]. In the ciliate Parameceum, guanylate cyclase is entirely results in autophosphorylation of the receptor as well as the membrane-bound [33]. The enzyme in the cilia is stimulated phosphorylation of some intracellular proteins. Further details on the mechanism of action are largely unknown. Some by Ca2+ via calmodulin that is tightly bound to the enzyme signal molecules operating via tyrosine kinase receptors may [34]. Intracellular Ca2+ concentrations fluctuate as a result of activate phosphatidylinositol-4,5-bisphosphate phospho- the C a 2 + / K + action potential which is evoked by various lipase C, possibly by phosphorylating the lipase at tyrosine stimuli 135,361. Thus, in Paramecium the Ca2+flux across the residues [8 - lo]. Tyrosine kinase receptor may also induce ciliary membrane is the first intracellular signal whereas the the phosphorylation of the protein that stimulates GTPase change of cGMP concentration is secondary [34]. In mamactivity of RAS, which may lead to the activation of another malian rod outer segments the cascade of events is just the unidentified transduction pathway [l 11. For the insulin recep- opposite, since the reduction of cGMP concentration is the tor, the stimulation of a specific phospholipase C generates a first intracellular event causing a change in Ca2+ concenwater-soluble inositol-containing phosphorylated polysac- tration [37, 381. The function of cGMP in Paramecium is charide. However, administration of this polysaccharide to unknown. In summary, some membrane-bound guanylate cyclases insulin-sensitive cells mimicks some but not all effects of insualso act as receptors (atrial natriuretic peptide in mammals lin in these cells, suggesting that it may mediate only a part of and rasact in sea urchin), whereas other membrane-bound insulin action [12 - 151. Many genes that encode tyrosine-kinase-possessing recep- enzymes appear to have no direct receptor function but are tors in mammalian cells have been cloned (see [7]). Similar activated more indirectly by Ca2+ (Paramecium), or by Greceptors with tyrosine kinase activity have been detected protein-coupled surface receptors ( D . discoideum). The reguin several invertebrates [7, 16, 171. However, no conclusive lation of soluble guanylate cyclase, which is present in virtually evidence is available about the existence of surface receptors all eukaryotes, is essentially unknown.

+

with tyrosine kinase activity in microorganisms. Cells of Neurospora crassa possess insulin binding activity and respond to insulin [18-201. In D. discoideum a small but significant amount of protein-derived phosphorylated amino acids is identified as phosphotyrosine (unpublished observations). It is not yet known whether these tyrosine residues are phosphorylated by enzymes that also possess receptor or signalling functions. The absence of more data on tyrosine kinases in microorganisms precludes a further discussion of this subject, but it may be expected that a search for homologous genes in microorganisms will establish whether this transduction pathway is present in all eukaryotes or only in the higher eukaryotes.

Sensory transduction by steroid-type receptors Most steroid-like hormones enter mammalian cells and bind to intracellular receptors. These receptors then adopt an activated conformation, allowing the receptor to bind to specific sequences that are present in the promotor region of target genes. Whether transcription is activated or not often depends on still other factors, such as cell-type-specific transcription factors [39 -431. Sensory transduction via steroid receptors is widespread in vertebrates and invertebrates; the occurrence in microorganisms is less well documented. Stereospecific and high-affin-

29 1 ity binding proteins for steroids have been identified and characterized in several eukaryotic microorganisms [44-481. Corticosterone and estrogen binding proteins have been found in the pathogenic yeast Candida albicans, estrogen binding proteins and an endogeneous ligand in the yeast Saccharomyces cerevisiae, estrogen binding and inhibition ofmyceliumto-yeast transformation in the fungus Paracoccidioides brasiliensis, and progesterone binding and inhibition of growth in the fungus Trichophyton metagrophytes. The mechanisms by which steroids function in microorganisms has been explored in the yeast S. cerevisiae: p-estradiol, a minor component of yeast cells, stimulates the recovery from growth arrest in early G1, probably by controlling the levels of CAMP by means of an increase of adenylate cyclase mRNA 1491. Further comparison of signal transduction via steroid receptors in vertebrates and microorganisms awaits complete characterization of steroid receptors in microorganisms, and establishment of binding activity to specific DNA sequences. Besides the steroid type of signal transduction, microorganisms may also possess non-steroid signal molecules with the accompanying receptors to regulate gene transcription. The mode of action of these molecules may resemble the way retinoic acid regulates development and differentiation [50]. In D. discoideum a signal molecule has been identified, called differentiation-inducing factor. It is a chlorinated diterpene, which induces stalk cell differentiation and the expression of stalk-cell-specific genes [51, 521. A cytosolic protein has been identified that binds this factor with high affinity and specificity [52a]and which may act like steroid receptors by binding to responsive elements on target genes. In summary, signal transduction via steroid receptors is present in vertebrates and in invertebrates. In eukaryotic microorganisms steroid receptors are present, but their mechanism of action is far from understood. Steroid hormones are generally not water-soluble; their mechanism of action in vertebrates depends on transport using a transport protein. It is possible that for this reason signal transduction via steroid hormones is present predominantly in multicellular organisms.

Sensory transduction by modulation of other transduction pathways

Some signal molecules modulate the transduction of other signal molecules. For instance, high concentrations of adenosine inhibit adenylate cyclase in mammalian cells and inhibit cAMP binding to surface receptors in D . discoideum [53 - 551. Both effects are mediated by the so-called P-site [56, 571. hi vertebrate cells, low concentrations of adenosine bind to specific adenosine receptors which interact with G, or Gi, the G-proteins that stimulate or inhibit adenylate cyclase, respectively [57]. Adenylate cyclase in these adenosine-responsive cells is also regulated by other hormones. In D. discoideum the mechanism of action of some signal molecules depends on the modulation of other signal transduction pathways. The morphogen differentiation-inducing factor also has short-term effects which appear to depend on the disruption of receptor-mediated adenylate cyclase activation, possibly by interference with G-protein functioning [58]. In D. discoideum, ammonia is involved in stalk cell differentiation. At least part of the mechanism of action of ammonia depends on the inhibition of CAMP-receptor mediated activation of adenylate cyclase [59].

Components of G-protein-coupled sensory transduction pathways Receptors coupled to G-proteins

The general structure of G-protein-coupled receptors consists of seven putative transmembrane-spanning domains; this hypothesis was derived from the identification of the encoding genes and deduced amino acid sequences [60 - 681. Receptors which bind small ligands, such as catecholamines and serotonin, have a small putative extracellular N-terminal domain. Receptors binding large hormones, such as luteinizing and thyroid-stimulating hormones, have large extracellular domains. It has been proposed that small ligands are bound in the barrier that is formed by the seven transmembrane domains whereas large ligands are bound to the extracellular N-terminal domain. The surface receptor of D. discoideum that binds the chemoattractant cAMP has the same proposed topology: a small extracellular N-terminal domain, seven putative transmembrane-spanning domains and a rather long cytoplasmic domain [30]. Three genes have been cloned that are homologous in the putative transmembrane-spanning domains and the connecting loops (Devreotes, Kimmel and Saxe, personal communication); however, the putative extracellular N-terminal domain and the cytoplasmic C-terminal domains are different. The divergence of the cytoplasmic domains is especially interesting, because one receptor contains multiple serines, which are absent in the other receptors. These serines are postulated to be heavily phosphorylated during desensitization as in p-adrenergic and rhodopsin receptors (see below). Mating in the yeast S. cerevisiae is induced by the a and a mating factors, small peptides that are secreted by a and a cells, respectively. The a mating factor binds to a receptors on a cells, whereas the a factor binds to a receptors on a cells. Activation of the receptors leads to morphological and developmental changes which prepare the cells for sexual conjugation [69 - 711. The a-factor and a-factor receptors are the product of the S T E 2 and STE3 genes. The predicted proteins have seven hydrophobic segments, and a putative cytoplasmic domain with multiple putative serine phosphorylation sites [72, 731. Persistent stimulation of cells with high ligand concentrations generally leads to desensitization. This may be mediated by several mechanisms, including uncoupling of surface receptors from transducing G-proteins, removal of the receptors from the cell surface (sequestration), degradation of the receptors (down regulation), regulation of receptor expression, and inhibition of down-stream effects [74,75]. These processes are often associated with ligand-induced phosphorylation of the receptor, which occurs in virtually all Gprotein-coupled receptors, including the P-adrenergic receptor [76, 771, the D. discoideum cAMP receptor [78-801 and the yeast a-factor and a-factor receptors [81]. Genetic and pharmacological manipulation of mammalian, D. discoideum and yeast cells suggest that receptor phosphorylation is not required for receptor sequestration, but is probably involved in receptor - G-protein uncoupling [81- 841. G-proteins

G-proteins are proteins that bind and hydrolyze GTP. At least three groups of G-proteins can be distinguished : large signal-transducing G-proteins such as G, and Gi, large Gproteins that do not transduce signals such as tubulin and

292

s'gr

fond

GDP/GTP exchange

GTPose octivaling protein

t

Signol 7 Hetero trimeric G-protein

Small G- protein

Fig. 2. The activation of signal-transducing G-proteins (left), and small G-proteins (right)

elongation factor Tu (which will not be discussed), and small G-proteins such as the protooncogene RAS [85]. Signal-transducing G-proteins. Signal-transducing G-proteins are heterotrimeric G-proteins that transduce the signal from a cell-surface hormone receptor to an intracellular effector protein. These G-proteins are composed of a 38 - 52-kDa M subunit, a 35 - 36-kDa p subunit and a 8 - 10-kDa y subunit [86 - 891. The c i subunit contains four functional domains for interaction with the f l y subunit, the receptor, the effector enzyme and guanine nucleotides, respectively (Fig. 2A). The GTP-binding site is formed by three structural domains which are nearly identical in all signal-transducing G-proteins, and conserved in all G-proteins [90,91]. These domains have been used to clone multiple genes that encode putative signal-transducing G-proteins [92]. In D. discoideum six genes have been cloned encoding five proteins that show strong homology with mammalian Gprotein M subunit, and one protein that is nearly identical to a G-protein p subunit. G,1 and G,2 were cloned by screening a cDNA library with oligonucleotides based on the sequence of the conserved GTP-binding domains [93]. Ga3was cloned by polymerase chain reaction using primers directed against these domains, and G,4 and G,5 were identified by low-stringency hybridization to a cDNA library of differentiated cells (Devreotes and Firtel, personal communication). Gal is expressed during growth, G,2 and G,3 during early and late cell aggregation, and G,4 and G,5 during multicellular differentiation. The gene encoding the /3 subunit is expressed during entire development [94] (and Devreotes, personal communication). Biochemical experiments supports the idea that multiple G-proteins are functional in D. discoideum; evidence has been presented for an adenylate-cyclase-stimulatory G-protein [95, 961, an adenylate-cyclase-inhibitory G-protein that is sensitive to pertussis toxin [96], and a G-protein that stimulates phospholipase C [97 - 991. The two putative G-proteins that regulate adenylate cyclase are probably not among the cloned Gal -G,5 proteins; it can not be excluded that these G-proteins belong to the class of small G-proteins (see below). The G-protein that regulates phospholipase C is most likely G,2; this conclusion is based on the observation that D. discoideum mutant f g d A [IOO] is defective in a G, subunit [101, 1021,

Table 1. The genetics of signal transduction in S.cereviciae In S . cereviciae strains, wild-type and mutant genes are indicated in capitals and lower case letters, respectively. PKA = protein kinase A Gene

Protein

STE2 STE3 G P A l or SCGl GPA2 STE4 STElX CYRl BC Y l TPK1,2,3 RASl,2 IRA1 CDC25

r-receptor a-receptor al-subunit G-protein a2-subunit G-protein j-subunit G-protein y-subunit G-protein adenylate cyclase regulatory subunit of PKA catalytic subunit of PKA the small G-protein RAS GTPase activating protein of RAS GDP/GTP exchange protein of RAS

the G,2 gene [94] and in GTP[yS]-mediated stimulation of phospholipase C [99] (and unpublished observations). The number and function of signal-transducing G-proteins in D . discoideum will be determined by modern molecular genetic techniques, identification of the genes by homology and inactivation of the genes by antisense mRNA inactivation or gene disruption [103, 1041. In yeast, multiple subunits of signal-transducing G-proteins have been cloned by reversed genetics or by cross hybridization with cDNAs of rat G, (Table 1). Two M subunits are encoded by the GPAI (also known as SCGI) and GPA2 gene [105-1071; the deduced amino acid sequences of the products of STE4 and STE18 genes show strong homology with the p subunit and weak homology with the y subunit of mammalian G-proteins, respectively [108]. The effector enzymes of these G-proteins have not been identified, but genetic experiments have unraveled much of their function [109]. The double mutant ste2 gpul has a very high mating efficiency, which is supressed by STE2, indicating that the GPAl G-protein functions downstream of the STE2 receptor [110]. Mutations in the CPAI gene lead to cell cycle arrest in G1, and imply that GPAl is involved in pheromone signal transduction, playing a role in mating [l 113. Null mutations

293 in either STE4 or STE18 suppress the mutation in GPAI, suggesting that the b) subunit initiates the pheromone response [108]. Over-expression of STE4 and STEI8 induce pheromone signal transduction, which is counteracted by over-expression of GPAI. This supports the idea that the py subunit is the active subunit [112]. GPA2 is probably not involved in pheromone signal transduction, because null mutants of GPA2 are viable, whereas over-expression of GPA2 leads to elevated cAMP levels [105]. Indirect evidence for the presence of signal-transducing Gproteins was obtained for various eukaryotic microorganisms using biochemical experiments. Stimulation of adenylate cyclase by GTP has been observed in N . crassa, S. cerevisiae, Phycomyces blukesleeunus, and D. discoideum [95, 96, 113 1151. In many microorganisms proteins have been identified that bind [35S]GTP[yS][116- 1191. These experiments do not necessarily imply the involvement of signal-transducing Gproteins, because many of the identified [35S]GTP[yS]-binding proteins have a molecular mass of 20 - 25 kDa and thus apparently belong to the class of small G-proteins. In some organisms, GTP[yS]-binding and GTPase activity are increased by receptor agonists, such as cAMP in D. discoideum [120, 1211 or light in invertebrate photoreceptors of Muscu, Culiphora, Octopus, squid, and Sepia [122- 1301, suggesting an interaction between a G-protein and a receptor. Receptorstimulated GTP-binding or GTPase may be considered a minimal criterium to propose the presence of signal-transducing G-proteins. Small G-proteins. Small G-proteins are probably not directly involved in the transduction of signals from surface receptors to effector enzymes. Small G-proteins are monomers of 17--25 kDa. They do not interact with the py subunit of signal-transducing G-proteins, and lack the two domains of signal-transducing G-proteins that are postulated to interact with the py subunit and the receptor, respectively [131, 1321. The family of small G-proteins consists of many members, including ras, ral, rab, YPT1, and SEC4 [133-1381. The function of many of these proteins is unknown, but some may be involved in organelle trafficking [I38 - 1471, while others, such as RAS, may indirectly interfere with signal transduction (see [132]). The mammalian protooncogene R A S encodes a 21-kDa GTP-binding protein; the oncogene form of the protein is generated by mutations in the GTP-binding domain that reduce GTPase activity and presumably lead to the constitutive activation of the protein (see [132]). The activity of RAS may be regulated by the GTPase activating factor (GAP) and a GDP-GTP exchange factor. The function of RAS is not known; RAS may regulate the activity of another, as yet undefined, effector enzyme, or GAP itself may be the effector enzyme [148]. Although GAP binds to the putative effector domain of RAS [148], expression of mammalian GAP in yeast does not suppress RAS mutations, suggesting that GAP is a modulator but not an effector of RAS [149]. In the yeast S. cerevisiae the activity of RAS is regulated by two other proteins that are the product of the genes CDC25 and IRA1 [150- 1541. The CDC25 protein is required to activate RAS proteins, probably by catalyzing the GDP-GTP exchange reaction [152]. Disruption of the I R A l gene suppresses the lethality of the cdc25 mutation, suggesting that the IRAl protein inhibits the function of the RAS proteins in a fashion antagonistic to the function of CDC25 protein [151]. IRAl protein shows amino acid sequence similarity with GAP [151, 3551, the GTPase-activating protein of the mammalian R A S protein, and may fulfill the same function for yeast RAS.

Recently proteins with functions similar to CDC25 and IRAl proteins were identified in mammalian cells [156- 1581, suggesting that other small G-proteins may also be regulated by a GDP - GTP exchange factor and by a GTPase-activating protein (see Fig. 2B).

Adenylate cyclase pathway The adenylate cyclase structural gene has been cloned from yeast and mammalian cells [159-1631. The deduced amino acid sequence of the mammalian enzyme resembles that of a transporter protein : twelve putative transmembrane-spanning domains are localized in two groups which are connected by a large 36-kDa cytoplasmic domain. The interconnecting domain is virtually repeated as a C-terminal cytoplasmic domain and shows strong similarity to domains in yeast adenylate cyclase and in vertebrate and sea urchin guanylate cyclase, suggesting that it is the catalytic domain [161]. In vertebrate cells, receptor stimulation of adenylate cyclase is mediated by the stimulatory G-protein, whereas inhibition is mediated via the inhibitory G-protein [164]. All these components have been purified and reconstitution of receptors, G-proteins and adenylate cyclase in lipid vesicles yields a hormone- and GTP-dependent adenylate cyclase activity [165, 1661. In contrast to verebrates and D. discoideum, yeast adenylate cyclase is stimulated by RAS [167, 1681. In S. cerevisiae, cAMP via the activation of protein kinase A has a crucial role in the cell cycle. cAMP produced by adenylate cyclase, encoded by the C Y R l gene [159], binds to the regulatory kinase subunit, encoded by the B C Y l gene [169]. This leads to the release of the catalytic subunit, which is encoded by T P K l , TPK2, or TPK3 genes [170]. Free catalytic subunits then phosphorylate target proteins whose phosphorylation is essential for cell cycle progression. Mutations that block the activation of protein kinase A, such as cyrl, cdc25, rasl ras2 or stel, prevent cells from entry into the mitotic cycle, whereas mutations that cause high levels of protein-kinase-A-dependent protein phosphorylation, such as bcyl, or iral, interfere with entry into the resting state [171]. Receptor-mediated activation of adenylate cyclase in D. discoideum is of special interest, because the receptor agonist is identical to the enzyme product: cAMP [172]. The cAMP produced by the cyclase is secreted by the cells and serves to activate the receptors on the same cells or on neighbouring cells [173- 1751.This autocatalytic feedback loop is controlled by an adaptation mechanism that interrupts the transduction of the cAMP signal from surface receptor to adenylate cyclase [176- 1781. When extracellular cAMP has been cleared by phosphodiesterase, the cells resensitize and a new burst of cAMP synthesis and secretion occurs. The sensory transduction pathway from surface receptors to adenylate cyclase in D. discoideum has not been completely identified. It is likely that stimulatory and inhibitory G-proteins are involved in this process, since GTP-mediated activation and inhibition of adenylate cyclase in a cell free system have been detected [95, 961. For maximal stimulation GTP[yS] must be present during cell lysis and the lysate must be incubated for an additional 5 min at 0°C before GTP[yS] mediated stimulation of adenylate cyclase can be detected [95]. Alternatively, stimulation by GTP[yS] can be observed if the adenylate cyclase assay with isolated membranes is performed at 0°C in the presence of a cytosolic protein [96]. This cytosolic protein appears to be essential for the loading of a G-protein with GTP, and is nonfunctional in the mutant SYNAG7 [95, 96, 1791. The protein

294 tivity during stimulation was found, suggesting multiple metabolic pools of PtdIns(4,5)P3 and/or Ins(1,4,5)P3 [207, 2081. It is possible that these different pools have different functions in cells. Ins(1 ,4,5)P3 induces a release of Ca2+ from non-mitochondrial pools which are presumably specific domains of the endoplasmatic reticulum [209]. The release of Ca2+ is mediated by an Ins(1,4,5)P3-stimulated Ca2+ channel. Recently the gene that codes for the putative Ins(1,4,5)P3 binding protein has been cloned, showing homology with the ryanodine receptor that releases calcium from sarcoplasmatic reticulum of skeletal muscle cells [210-2351. The detection of Ca2+ levels in single cells has altered our view on the role of Ins(1,4,5)P3 in Ca2+ release, because the increase of intracellular Ca2+ is not gradual, but shows oscillations whose frequency depends on the concentration of the hormone [216- 2241. It is presently unclear how these oscillations are generated. Many of the features described above for vertebrate cells are also found in D. discoideum; however, some aspects are very different (Fig. 3). The receptor agonist cAMP and the G-protein agonist GTP[yS] stimulate the formation of Ins(1,4,5)P3 [97, 981. The concentration of Ins(1,4,5)P3 increases from about 3.3 pM to 5.5 pM [206]. There is a substantial secretion of Ins(1,4,5)P3 in D.discoideum [206]; this may also occur in other microorganisms such as yeast and Paramecium, but seems to be absent in mammalian blood cells (unpublished observations). In Dictyostelium, the G-protein that transduces the receptor-mediated stimulation of Ins(1 ,4,5)P3 in D. discoideum has been tentatively identified as the Ga2 gene product. This conclusion is based on the observation that the D. discoideum mutant JgdA lacks this Ga2 gene product as well as GTP[yS]-mediated stimulation of Ins(1,4,5)P3 formation [94, 99, 1011. D. discoideum sheds a special light on the presumed role of Ins(1,3,4,5)P4 for Ca2+ Inositol phosphate/Cu2 'puthwuy uptake by the cell or Ca2' sequestration [225-2301: Ca2+ In vertebrates, stimulation of a specific phospholipase C uptake and sequestration are present in D. discoideum, but leads to hydrolysis of phosphatidylinositol 4,5-bisphosphate Ins(1,3,4,5)P4 is apparently absent in this organism (see be[PtdIns(4,5)P2] into diacylglycerol and inositol 1,4,5- low). In the yeast S. cerevisiae, phosphoinositol turnover is regutrisphosphate [Ins(1,4,5)P3]. Ins(1 ,4,5)P3 releases Ca2+ from a non-mitochondria1 pool, whereas diacylglycerol and Ca2+ lated by glucose [231, 2321. Glucose depletion induces stimulate the calcium/phospholipid dependent protein kinase S. cerevisiue cells to arrest in GO/Gl phase of the cell cycle. C [200- 2021. Calcium may also stimulate calmodulin-depen- When glucose is added back to these cells, an elevation of 32P dent enzymes and other calcium-dependent activities such as incorporation into phosphatidic acid, PtsIns, PtdInsP, and the polymerization of actin. The regulation of the inositol PtdInsP2 is observed. In [3H]inositol-labelled cells glucose cycle/Ca2 pathway by hormones and intracellular factors is induces the accumulation of InsP and InsP3. Furthermore, an antibody against PtdIns(4,5)P2, when introduced into yeast expected to be essential for many cellular activities. Function of the inositol cycle/Ca2+ pathway. Many hor- cells, induces growth arrest [233]. Temperature sensitive mumones stimulate the production of [3H]Ins(1,4,5)P3 in ver- tants for growth were isolated which show a different sensitebrate cells that are labelled with [3H]inositol. The increase tivity to this antibody (mutants piml -pim5). In piml and is usually rapid with a maximum at a few seconds after stimu- pim2 mutants, PtsIns(4,5)P2 or Ins(1 ,4,5)P3 together with dilation; the stimulation varies from barely detectable in one acylglycerol stimulate growth at the restricted temperature. system to as much as 50-fold in another system (see [201]). The Piml mutant has low PtdIns kinase activity, whereaspim2 Stimulation of Ins(1 ,4,5)P3 production can also be provoked mutant shows low PtdIns4P kinase activity; both mutants by GTP[yS] in cell-free systems or in permeabilised cells, have reduced levels of PtdIns(4,5)P2 at the restricted temperasuggesting that stimulation of phospholipase C is mediated ture. These data suggest thatpiml andpim2 could be the genes for PtdIns and PtdIns4P kinase respectively, and that the by a G-protein [203, 2041. The Ins(1,4,5)P3 response has also been measured with an production and breakdown of PtdIns(4,5)P2 may be essential Ins(l,4,5)P3-binding protein, which allows detection of the to proceed through the G1 stage in the yeast cell cycle [233]. Since both cAMP and inositol phosphates play an essential mass of Ins(1,4,5)P3 [205, 2061; in most experiments the increase of radioactivity and mass of Ins(l,4,5)P3 show similar role in the yeast cell cyle, it is intriguing to analyse the inositol kinetics, but the relative increase of the mass is sometimes cycle in cAMP mutants [234]. Mutants with low cAMP levels smaller than the increase of radioactivity. In some exper- (rus2, cyrl-2) have low PtdIns and PtdIns4P kinase activity, iments, in which the mass and radioactivity of Ins(1,4,5)P3 which is increased strongly upon addition of cAMP to these were measured simultaneously, an alteration of specific ac- cells. The activity of these kinases is very high in bey1 mutants,

shows some functional similarities with the GDP - GTP exchange factor CDC25 and the GTPase activating factor IRA1 of S. cerevisiae RAS [179]. In D. discoideum not all the CAMP produced is secreted. A small portion may bind to intracellular receptor proteins : the CAMP-dependent protein kinase A [180- 1821 and the recently identified CAMP-binding protein CABP-1 [183, 1841. The gene that codes for the regulatory subunit of protein kinase has been cloned; the deduced amino acid sequence predicts a protein that is homologous to the mammalian protein kinase, and different from the surface cAMP receptor [185]. The strong homology between vertebrate and D. discoideum protein kinase A is also reflected in the nearly identical cyclic-nucleotide-binding specificity of these proteins [186]. The function of the cAMP signal transduction pathway in D.discoideum has been investigated using several different approaches, including cAMP derivatives that specifically activate either surface cAMP receptors or intracellular protein kinase A [29, 187-1931, mutants that are defective in the activation of adenylate cyclase (SYNAG7) [194, 1971, transformants that over-express the regulatory subunit of protein kinase A [195], or transformants that express antisense mRNA complementary the the mRNA encoding the surface cAMP receptor [30]. These studies suggests that all functions of extracellular CAMP are mediated by surface receptors. The activation of adenylate cyclase is not required for cell growth, chemotaxis and differentiation, but is required for the production of the extracellular relay signal and for the fine regulation of the developmental program. Intracellular cAMP apparently has a function in many eukaryotic microbes, because CAMP-dependent protein kinases have been demonstrated in S. cerevisiue, Mucor rouxii, and Tripanommu cruzi [169, 170, 196-1991,

+

295 which have a constitutive activated protein kinase A. These results suggest that CAMP-dependent phosphorylation enhances poly(phospho)inositol lipid synthesis through the activation of PtdIns and PtdIns4P kinase activity, an effect which may lead to the enhanced production of Ins(1,4,5)P3 and diacylglycerol. i n conclussion, the inositol-phospholipid-derived second messengers Ins(1,4,5)P3 and diacylglycerol play important second messenger functions, not only in vertebrate hormone action, but also in invertebrate vision [235, 2361, and in the microorganisms D. discoideum and S . cerevisiae. Metabolism of inositolphospholipids. In vertebrates, inosito1 and CDP-diacylglycerol are converted to PtdIns, which is subsequently phosphorylated to PtdIns4P and PtdIns(4,5)P2. These phospholipids are hydrolyzed by phospholipase C activity 12371; the products of these reactions are InslP, Ins(1,4)P2and Ins(1 ,4,5)P3, respectively. Recent experiments [238- 2441 point to a more complex metabolism of the inositol phospholipids. Various isomers were found, such as PtdIns3P derived from PtdIns, PtdIns(3,4)P2 presumably derived from PtdIns4P, and a PtdInsP3 isomer with the putative isomeric structure PtdIns(3,4,5)P3. The function of these inositol phospholipids that are phosphorylated at the 3-position is unclear at present, but they could be important for the transduction of mitogenic and oncogenic signals. These phospholipids are not substrates of the phospholipase C activities that have so far been purified [244, 2451. It is possible that these compounds are substrates of other enzymes; interesting candidates are phospholipase D and phospholipase A2 [246, 2471 which would yield the highly mitogenic lysophosphatidic acid [248] as product. The metabolism of inositol phospholipids has been less well defined in microorganisms than in vertebrates. Remarkably, a phospholipase C activity has not been demonstrated in D. discoideum lysates [249]. However, based on the kinetics of label incorporation of [32P]orthophosphate and [3H]inositol in D. discoideum cells, PtdIns, PtdInsP and PtdInsP2 were identified [97]. The polar head groups of the lipids were not identified. In membranes, the kinases that phosphorylate PtdIns and PtdIns4P were characterized, but again the polar head groups of the products were not identified [250]. Furthermore, the fatty acid composition of inositol-containing phospholipids should also be determined, because D. discoideum is reported to be devoid of arachidonic acid [251]. Inositol-containing phospholipids from Paramecium are also devoid of arachidonic acid [252]. Metabolism of inositol phosphates. In vertebrates Ins(1,4,5)P3 is metabolized by two routes: direct dephosphorylation to inositol, or phosphorylation to Ins(1,3,4,5)P4 followed by a complex series of dephosphorylations and phosphorylations [201, 202, 253, 2541. Ins(1,4,5)P3 is degraded to ins(l,4)P2 by an Ins(1,4,5)P3 Sphosphatase [255]. The enzyme activity is predominantly membrane-bound, although two soluble iso-enzymes have been identified in brain tissue [256, 2571. These iso-enzymes have different substrate specificity with respect to Ins(l,4,5)P3 and lns(1 ,3,4,5)P4. The kinase that phosphorylates Ins(1,4,5)P3 at the 3-position is present in a soluble form in virtually all mammalian cells [229, 258, 2591 and is activated in most if not all tissues by Ca2+/calmodulin[260,261]. Moreover, Ins(1,4,5)P3 3-kinase and 5-phosphatase activities may be stimulated by agents that activate protein kinases A and C, respectively [262, 2631. Furthermore, Ins(1,3,4,5)P4 is a potent inhibitor of the Ins(1,4,5)P3 5-phosphatase [257]. Thus, these two Ins(1 ,4,5)P3-metabolizing enzymes are controlled

by various mechanisms, which determine the kinetics of the Ins(1,4,5)P3 and Ins(1,3,4,5)P4 response, as well as the relative concentration of these two compounds. Even though Ins(1,3,4,5)P4 may not have second messenger functions, its formation strongly controls Ins(1,4,5)P3 levels. Both ins(1,3,4)P3 and Ins(1,4)P2 are substrates of the same l-phosphatase; Ins(1,4,5)P3 is not degraded by this enzyme [264 2661. The Ins(1,3,4)P3/Ins(1,4)P2 1-phosphatase, as well as the InsP monophosphatase, are Li+ sensitive enzymes [267, 2681. Finally, Ins(1,3,4)P3 can be phosphorylated to Ins(1,3,4,6)P4, which could be the pathway for InsP, synthesis [254]. The similarities and differences of the metabolism of Ins(1,4,5)P3 in D. discoideum and vertebrates are indicated in Fig. 3. D . discoideum cells possess the Ins(1,4,5)P3 5-phosphatase, the Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase and the InsP monophosphatase [269]. However, D. discoideum cells apparently do not have the Ins(1 ,4,5)P3 3-kinase activity, and therefore the complex metabolism of Ins( 1,3,4,5)P4 is virtually absent in D. discoideum [97]. Remarkably, the various functions that have been attributed to Ins(1,3,4,5)P4 in vertebrate cells, such as calcium uptake and calcium sequestration, are performed in D. discoideum without the presence of Ins(1,3,4,5)P4. Although 1ns(1,4,5)P3 is dephosphorylated in D. discoideum to inositol by the vertebrate route via Ins(1 ,4)P2 and Ins4P, this route is of minor importance in aggregating D. discoideum cells since Ins( 1,4,5)P3 is dephosphorylated mainly at the I-position to Ins(4,5)P2 and subsequently to Ins4P [269]. The presence of multiple dephosphorylation routes in D . discoideum suggests that these routes are regulated and that the InsP2 products may have specific functions. Indeed, the metabolism of Ins(1 ,4,5)P3 changes dramatically during differentiation of D. discoideum cells (unpublished observations). The metabolism of inositol phosphates in other microorganisms has not been studied in detail, but seems to proceed as in vertebrates. In plant cells the metabolism of Ins(1,4,5)P3 might occur as in D. discoideum yielding Ins(1,4)P2, Ins(4,5)P2, Ins4P and inositol [270]. In contrast to D . discoideum, none of the inositol-phosphate phosphatases in plant cells are lithium-sensitive; furthermore, Ins(1,3,4)P3 has been detected in plant cells, suggesting the presence of the Ins(1,4,5)P3 3-kinase [271]. Lithium ions inhibit the activity of two vertebrate enzymes, the inositol monophosphate phosphatase and the inositol polyphosphate 1-phosphatase that dephosphorylates Ins(1 ,4)P2 and Ins( 1,3,4)P3 (see [272]). Consequently, the recycling of inositol as well as the de novo synthesis from glucose 6-phosphate are inhibited, and cells rely on the uptake of extracellular inositol for replenishment of hydrolysed inositol phospholipids. Thus lithium ions will have a more profound effect on cells which can not recruit inositol from the extracellular medium, such as brain cells. Furthermore, since lithium ions are uncompetitive inhibitors of the phosphatases, the inhibition of inositol recycling is far more pronounced in cells with a very active inositol cycle, than in more quiescent cells. These effects of lithium are combined in the ‘inositol depletion theory’ and have been used to explain the pharmacological effect of lithium treatment of manic depressive desease : the over-active cells are damped by inositol depletion whereas the more quiescent cells are not affected [272]. This attractive hypothesis is somewhat complicated by observations that lithium ions also interfere with receptor - G-protein interactions in the rat cortex [273].

296

Fig. 3. The inositol cycle ofmammalian and D. discoideum cells. D. discoideum cells do not have the kinase that phosphorylates Ins(1,4,5)P3, and has two enzymes constituting an additional dephosphorylation pathway through Ins(4,5)]P2. DG, diacylglycerol; PA, phosphatidic acid

Lithium ions have profound effects on the development of D. discoideum cells : lithium ions promote prestalk differentiation and inhibit prespore differentiation [274, 2751. The effects of lithium on inositol phosphate phosphdtases in D. discoideum is similar to the effects in mammalian cells with the exception that D. discoideurn cells contain an additional enzyme that is lithium sensitive: the Ins(1 ,4,5)P3 l-phosphatase [269]. The overall effect of lithium treatment is a reduction of the mass of Ins(1,4,5)P3 [275]. Functiorz o j protein kinase C. The stimulation of phospholipase C does not only lead to the production of inositol phosphates but also of diacylglycerol. Recent experiments suggest that the initial increase of diacylglycerol may be derived from phospholipase-C-mediated inositol phospholipid degradation, whereas the prolonged accumulation of diacylglycerol is derived from the degradation of other phospholipids, notably phosphatidyl choline [247]. Diacylglycerol and Ca2 stimulate protein kinase C. Different protein kinase C isoenzymes have been identified that are coded by different genes. These isoenzymes show different sensitivity to the activators diacylglycerol, CaZ , phosphatidylserine and the artificial activator phorbol esters. It is likely that the isoenzymes will also show different substrate specificities [200]. In D. discoideum an enzyme activity like protein kinase C was recently identified [276]. The existence of this enzyme was predicted by the effects of phorbol ester/ATP/Ca2 on CAMPbinding to surface receptors [277,278]. It has also been reported that phorbol esters, in combination with Ins(1,4,5)P3, induces prespore gene expression in saponin permeabilized D. discoidezim cells [279]. A more complete picture of the function of protein kinase C in D . discoideum awaits the cloning of the encoding gene(s) and the reduction of transcription by antisense mRNA inactivation or gene disruption. Enzymes like protein kinase C are probably present in various lower eukaryotes, for instance the sponge Geodia cydonium [280]. Function ?If RAS in inositol phosphate signalling. In yeast, RAS directly regulates the activity of adenylate cyclase [2811; the effects of RAS on the poly(phospho)inositol lipid turnover may be indirect as was described above [234]. No other organisms have been found in which RAS affects the activity of adenylate cyclase directly [282, 2831. It has been suggested +

+

+

that RAS interacts with the inositol cycle, presumably by activating phospholipase C. In some cells that are transformed with the RAS oncogene it was observed that the levels of Ins(1,4,5)P3 were increased [284, 2851, but this appears not to be a general phenomenon [286-2881. In addition, the activation of protein kinase C, which is repeatedly observed in RAS-transformed cells [289,290], is not always detectable and thus cannot generally explain the transformed phenotype [291, 2921. The collected data suggest that RAS may interact with some step in the inositol cycle in some cells, but the effect, whenever present, is probably indirect and may not be responsible for the oncogenic transformation. D. discoideum cells contain two genes that are highly similar to the mammalian RAS genes, one gene expressed during growth [293] and one during differentiation [294]. Cell lines have been constructed that over-express either the wildtype RAS gene or a RAS gene encoding a protein with a Gly 12-+Thr mutation which would confer oncogenic activity in mammalian RAS genes [295]. Over-expression of the Thrl2coding RAS gene produces a biochemical phenotype that is consistent with activation of the inositol phosphate/Ca2+/ protein kinase C pathway [278, 296, 297, 2981: Ins(l,4,5)P3 levels are increased as well as intracellular CaZ+levels; as a result, guanylate cyclase is more rapidly inactivated by Ca2 after receptor-mediated activation. Also putative protein kinase C is activated. A more detailed investigation of the inositol cycle suggests that increased Ins(1 ,4,5)P3levels in the mutated RAS transformant is not due to increased phospholipase C activity, but to the enhanced conversion of PtdIns to PtdInsP, leading indirectly to increased levels of PtdInsPz and Ins(1 ,4,5)P3 (unpublished observations); the reason why PtdIns kinase activity is increased in these RASThrl2 protein transformants is presently unclear. In summary, an inositol cycle as known in vertebrates appears to exist also in eukariotic microorganisms as well as in plant cells. In none of the systems it is precisely known how surface receptors activate phospholipase C. Ins(1 ,4,5)P3 may be metabolized by 1-phosphatase, 5-phosphatase or 3-kinase in a highly regulated order. The metabolism of Ins(1,4,5)P3, as well as its regulation, may differ in different organisms. Finally, other phospholipids and phospholipase activities are expected to play a role in sensory transduction as well. +

297 Ion channels In vertebrates a number of ion channels are regulated by surface receptors (see [299- 3021). G-proteins are involved in the regulation of some channels. Thus, hormonal inhibition of Ca2'-channels in neuronal and endocrine cells is mediated by a pertussis-toxin-sensitive G-protein. A cholera toxin-sensitive G-protein may stimulate cardiac Ca2+channels without the involvement of a CAMP-dependent intermediate. K + channels are opened by a pertussis-toxin-sensitive G-protein in mammalian cells. Other ion channels are regulated by cyclic nucleotides, such as the opening of the voltage-dependent Na+ channel by cGMP in the retina. Finally, the dihydropyridinesensitive Ca2+ channel from skeletal muscle is regulated by phosphorylation by protein kinase A, whereas cGMP-dependent protein kinase (protein kinase G) increases the activity of the voltage dependent Ca2+channel in snail neurons. Thus, some channels are regulated directly by ligand, others by Gprotein subunits, and still others by second messengers or kinases. In D. discoideum cells the regulation of ion channels by extracellular signals is only partly understood. A major problem is that the composition of the plasma membrane apparently prevents the formation of a tight seal required for pathclamp analysis [303]. It is known, however, that extracellular cAMP increases the efflux of H i and K + and the influx of Ca2+ [304-3081. The transport of protons is most likely mediated by an electrogenic H +/ATPase and appears to be the main determinant of the plasma membrane potential [309]. K + transport is probably regulated by intracellular Ca". The transport of Ca2+ over the plasma membrane is regulated by a Ca'+/ATPase that pumps Ca2+ out of the cell and by a Ca2+ channel that is opened by receptor agonists and leads to the entrance of Ca'' [310-3141. The sensory transduction pathway from surface receptors to the ion channels is essentially unknown. The importance of all these ion translocations for cellular functions, however, is very limited. Removal of extracellular Ca2+ with EGTA does not interfere with chemotaxis, cell aggregation and differentiation [315,316].Moreover, signal transduction and chemotaxis are essentialy normal in electropermeabilized cells, in which free diffusion of ions and small metabolites through the plasma membrane is made possible [97]. Using this method, it was established that the membrane potential and the translocation of K f and N a + are not important for signal transduction and chemotaxis in D. discoideum [317]. It is likely that intracellular Ca2+is more important, because electroporated D. discoideum cells in the presence of EGTA no longer respond chemotactically to cAMP (B. Van Duijn and P.J.M. Van Haastert, unpublished observations). The relative unimportance of ions and membrane potential in D. discoideum signal transduction is different from the role in other microorganisms such as Paramecium [318]. In the latter organism a C a 2 + / K +action potential that is evoked by various stimuli plays a central role in signal transduction [35, 361. Interaction of signal transduction pathways

The signal transduction pathways that have been described above do not operate independently, but affect each other. The inhibitory G-protein, as well as surface receptors that are coupled to the adenylate-cyclase-stimulating G-protein, are substrates of protein kinase C; phospholipase C may be phosphorylated by tyrosine kinase activity associated with growth

factor receptors and the epidermal-growth-factor-induced hydrolysis of phospholipids is modulated by protein kinase C 13191. On the other hand, some responses, such as neuronal induction, are more potently activated if two signal transduction pathways (protein kinase A and C) are stimulated simultaneously [320]. Also, the response that is induced by a stimulus may depend on the way the stimulus is applied, e.g. in D. discoideum pulses of nanomolar cAMP or the addition of constant micromolar CAMPinduces the expression of different genes [321]. This complex network of interactions is probably one of the more difficult problems to be understood. The genetics of signal transduction in D. discoideum

In D. discoideum the genetics of signal transduction has been investigated using several mutants that are defective in specific components of the network (Fig. 4). All signal transduction depends on the presence of surface cAMP receptor as was established by studies with cells in which the expression of the receptor gene was inactivated [30]. Three mutants have been used to investigate the role of the adenylate cyclase pathway. In mutant pdsA extracellular phosphodiesterase activity is absent [322], in mutant HB3 cAMP secretion is impaired [323, 3241, and in mutant SYNAG7 the G-proteinmediated activation of adenylate cyclase is lost [95,961. The activation of guanylate cyclase and phospholipase C are essentially normal in these mutants, and CAMP induces normal chemotaxis and cell differentiation [95, 96, 324, 3251. This suggests that the main function of the activation of adenylate cyclase is to generate the extracellular cAMP relay signal. The function of intracellular cGMP has been investigated using the mutant stmFwhich appears to be defective in cGMP phosphodiesterase [326- 3281. In this mutant extracellular CAMPinduces elevated and prolonged cGMP accumulation, which is associated with prolonged chemotactic movement. The enhanced accumulation of cGMP in mutant stmF has no effect on the stimulation of adenylate cyclase or phospholipase C (unpublished observations). The importance of the cGMP pathway will be established more accurately when mutants lacking guanylate cyclase are available. Activation of the inositol cycle appears to be essential for all sensory transduction as was established using mutant f g d A which appears to be defective in the phospholipase-cactivating G-protein [99-1011. It is not yet known which components of the inositol phosphate/Ca2 pathway are essential for the activation of adenylate cyclase and guanylate cyclase. More mutants in the inositol phosphate/Ca' + pathway are strongly required to unravel the functions of the many components of the inositol cycle. +

A comparison of signal transduction between eukaryotic microorganisms and vertebrates The previous description of sensory transduction in different organisms leads to several conclusions. Essentially all sensory transduction mechanisms known from vertebrate cells are also present in invertebrates; however, in eukaryotic microbes several sensory transduction pathways are not conclusively identified. These pathways include sensory transduction via steroid receptors or via receptors with tyrosine kinase activity. Still, the similarity of signal transduction via G-protein coupled receptors in eukaryotic microorganisms and vertebrates is the most remarkable notion of the present comparison. Although the evolutionary distance between D. dis-

298

I -CAMP

c

~

~I

i \I

DAG

P

PLC /

d

\

IP33,\ip3-

function ?

-

c2+

aclin

/

\

\ / myosin

polymcrimtionj

...

S’AMP

cGMP

I

chemotaxis

\ SGMP

Fig. 4. Modelfor sensorj>transduction in Dictyostelium. CAMP may bind to two forms of surface receptors that activate the relay pathway (RA)and the chemotaxis/differentiation pathway (RB),respectively. The relay pathway is composed of unidentified G-proteins with stimulatory and inhibitory properties, GRP (an associated protein defective in mutant SYNAG7), and adenylate cyclase. cAMP is secreted by an unknown mechanism that is defective in mutant HB3. The chemotaxis/differentiation pathway is composed of the G-protein G,2, guanylate cyclase and phospholipase C. The second messengers cGMP and diacylglycerol/Ins(l ,4,5)P3/Ca2+ are involved in actin and myosin polymerization, resulting in chemotaxis. It is likely that diacylglycerol and Ca2+,either alone or in combination, are involved in certain types of gene expression. Several cross-interactions also exists, notably the inhibition of guanylate cyclase by C a Z f and the requirement of the B pathway for the activation of adenylate cyclase. Several mutants are indicated in boxes. PDE, cyclic nucleotide phosphodiesterase; DAG, diacylglycerol; AdCy, adenylate cyclase; GuCy, guanylate cyclase; PLC, phospholipase C; IP3, Ins(1,4,5)P3; GRP, GTP reconstituting protein.

coideum and vertebrates is more than a billion years, surface receptors, G-proteins, adenylate cyclase, guanylate cyclase and the inositol cycle are remarkably well conserved [3293321. D. discoideum surface cAMP receptors and yeast mating factor receptors have the typical structure of seven putative spanning domains and a long C-terminal cytoplasmic domain with multiple phosphorylation sites; this topology is essentially identical to vertebrate G-protein-coupled receptors, including adrenergic and muscarinic receptors. The powerful genetic techniques that can be used in microorganisms are proving to be very useful to study sensory transduction in these organisms. In the yeast S. cerevisiae many components of the sensory transduction pathways were identified by a combination of biochemistry and molecular genetics. D. discoideum has the additional advantage of a tightly programmed development of identical free moving amoeboid cells to a multicellular organism consisting of two or three cell types [333, 3341. It is expected that in the coming years we may learn in much more detail how simple extracellular signals regulate complex cellular functions such as chemotaxis and cell differentiation.

REFERENCES 1. Alberts, R . , Bray, D., Lewis, J., Raff, M., Roberts, K. &Watson, J. D. (1 989) Molecular biology of the cell, pp. 682 - 726, Garland Publishing Inc.. New York & London. 2. Casey, P. J. & Gilman, A . G. (1988) J . Biol. Chem. 263,25772580.

3. Lefkowitz, R. J. & Caron, M. G. (1988) J . Bid. Chem. 263, 4993 - 4996. 4. Dohlman, H. G., Caron, M. G. & Lefkowitz, R. J. (1987) Biochemistry 26, 2657 - 2664. 5. Hunter, T. (1987) Cell50, 823-829. 6. Williams, L. T. (1989) Science 243, 1564- 1570. 7. Hunter, T. (1989) Cell58, 1013-1016. 8. Nishibe, S, Wahl, M. I., Rhee, S. G. & Carpenter, G. (1989) J . B i d . Chem. 264, 10335-10338. 9. Meisenhelder, J., Suh, P. G., Rhee, S. G. & Hunter, T. (1989) Cell57, 1109-1122. 10. Margolis, B., Rhee, S. G., Felder, S. Mervic, M., Lyall, R., Levitzki, A., Ulldch, A,, Zilberstein, A. & Schlessinger, J. (1989) Ce1157, 1101-1107. 11. Molloy, C. J., Bottaro, D. P., Fleming, T. P., Marshall, M. S., Gibbs, J. B. & Aaronson, S. A. (1989) Nature 342, 711 -714. 12. Low, M. G. & Saltiel, A. R. (1988) Science 239, 268-275. 13. Czech, M. P., Klarlund, J. K., Yagaloff, K. A,, Bradford, A. P. & Lewis, R. E. (1988) J. Biol.Chem 263, 11017-11020. 14. Gaulton, G. N., Kelly, K. L., Pawlowski, J., Mato, J. M. & Jarett, L. (1988) Cell 53, 963-970. 15. Villalba, M, Kelly, K. L. & Mato, J. M. (1988) Biochim. Biophys. Acta 968, 69 - 76. 16. Basler, K. & Hafen, E. (1988) Trends Genet. 4, 74-79. 17. Casanova, J. & Struhl, G. (1989) Genes Dev. 3.2025-2038. 18. McKenzie, M. A., Fawell, S. E., Cha, M. & Lenard, J. (1988) Endocrinology 122, 511 -517. 19. Dietz, E., Uhlenbruck, G. & Lutticken, R. (1989) Naturwissenschuften 76, 269 - 270. 20. Fawell, S. E. & Lenard, J. (1988) Biochem. Biophys. Res. Commun. 155, 59-65. 21. Garbers, D. L. (1989) J . Biol. Chem. 264, 9103-9106.

299 22. Thorpe, D. S. & Garbers, D. L. (1989) J . Biol. Chem. 264,6545 6549. 23. Lowe, D. G., Chang, M. S., Hellmiss, R., Chen, E., Singh, S., Garbers, D. L. & Goeddel, D. V. (1989) EMBO J . 8, 13771384. 24. Chang, M. S., Lowe, D. G., Lewis, M., Hellmiss, R., Chen, E. & Goeddel, D. V. (1989) Nature 341,68-72. 25. Chinkers, M., Garbers, D. L., Chang, M. S., Lowe, D. G., Chin, H., Goeddel, D. V. & Schulz, S. (1989) Nature 338, 78-83. 26. Chinkers, M. & Garbers, D. L. (1989) Science 245, 1392- 1394. 27. Mato, J. M., Krens, F. A., Van Haastert, P. J. M. & Konijn, T. M. (1977) Proc. Natl Acad. Sci. USA 74,2348-2351. 28. Wurster, B., Schubiger, K., Wick, U. & Gerisch, G. (1977) FEBS Lett. 76, 141- 144. 29. Van Haastert, P. J. M. & Kien, E. (1983) J . Biol. Chem. 258, 9636 - 9642. 30. Klein, P. S., Sun, T. J., Saxe 111, C. L., Kimmel, A. R., Johnson, R. L. & Devreotes, P. N. (1988) Science 241, 1467- 1472. 31. Janssens, P. M. W., De Jong, C. C. C., Vink, A. A. & Van Haastert, P. J. M. (1989) J. Biol. Chem. 264,4329-4335. 32. Koch, K. W. & Stryer, L. (1988) Nature 334,64-66. 33. Klumpp, S. & Schultz, J. E. (1982) Eur. J . Biochem. 124, 317324. 34. Schultz, J. E., Pohl, T. & Klumpp, S. (1986) Nature 322, 271 273. 35. Kung, C. & Saimi, Y. (1985) Curr. Top. Membr. Transp. 23,4566. 36. Saimi, Y., Hinrichsen, R. D., Forte, M. & Kung, C. (1983) Proc. Natl Acad. Sci. USA 80, 5112-5116. 37. Stryer, L. & Bourne, H. R. (1986) Annu. Rev. Cell Biol. 2, 391 419. 38. Stryer, L. (1986) Annu. Rev. Neurosci. 9, 87 - 119. 39. Beato, M. (1989) Cell 56, 335 - 344. 40. Berg, J. M. (1989) Cell 57, 1065-1068. 41. Green, S. & Chambon, P. (1988) Trends Genet. 4, 309-313. 42. Ptashne, M. (1988) Nature335,683-689. 43. Levine, M. & Manley, J. L. (1989) Cell59,405-408. 44. Skowronski, R. & Feldman, D. (1989) Endocrinology 124, 1965- 1972. 45. Loose, D. S., Schurman, D. & Feldman, D. (1981) Nature 293, 477 - 479 46. Feldman D., Do, Y., Burshell, A,, Stathis P. & Loose D. S. (1982) Science 218,297-298. 47. Loose, D. S., Stover, E. P., Restrepo, A., Stevens, D. A. & Feldman, D. (1983) Proc. Nut1 Acad. Sci. USA 80, 76597663. 48. Schar, G., Stover, E. P., Clemons, K. V. Feldman, D. & Stevens, D. A. (1986) Infect. Zmmunol. 52, 763. 49. Tanaka, S., Hasegdwa, S., Hishinuma, F. & Kurata, S. (1989) CeN57, 675-681. 50. De The, H., Del Mar Vivanco-Ruiz, M., Tiollais, P., Stunnenberg, H. & Dejean, A. (1990) Nature 343, 177-180. 51. Morris, H. R., Taylor, G. W., Masento, M. S., Jermyn, K. A. & Kay, R. R. (1987) Nature 328, 811 -814. 52. Kay, R. R., Berks, M., Traynor, D., Taylor, G. W., Masento, M. S. & Morris, H. R. (1988) Dev. Genet 9, 579 - 587. 52a. Insall, R. Kay, R. R. (1990) Embo J . 9,3323-3328. 53. Newell, P. C. & Ross, F. M. (1982) J. Cen. Microbiol. 128, 2715 - 2724. 54. Theibert, A. & Devreotes, P. (1984) Dev. Biol. 106, 166-173. 55. Van Haastert, P. J. M. (1983) J. Bid. Chem. 258, 9643 -9648. 56. Van Lookeren Campagne, M. M., Schaap, P. & Van Haastert, P. J. M. (1986) Dev. Bzol. 117,245-251. 57. Wolff, J. Londos, C. & Cooper, D. M. F. (1981) Biosci. Rep. 3, 309 - 322. 58. Wang, M., Van Haastert, P. J. M. & Schaap, P. (1986) Differentiation 33, 24-28. 59. Williams, G. B., Elders, E. M. & Sussman, M. (1984) Dev. Biol. 105, 377 - 388. 60. Dixon, R. A. F., Kobilka, B. K. Strader, D. J., Benovic, J. L., Dohlman, H. G., Frielle, T., Bolanowski, M. A., Bennett, C. D., Rands, E., Diehl, R. E., Mumford, R. A., Slater, E. E.,

Sigal, I. S., Caron, M. G., Lefkowitz, R. J. & Strader, C. D. (1986) Nature 321, 75-79. 61. Emorine, L. J., Marullo, S., Briend-Sutren, M. M., Patey, G., Tate, K., Delavier-Klutchko, C. & Strosberg, D. A. (1989) Science 245, 1118-1121. 62. Liao, C. F., Themmen, A. P. N., Joho, R., Barberis, C., Birnbaumer, M. & Birnbaumer, L. (1989) J . Biol. Chem. 264, 7328-7337. 63. Kubo, T., Fukuda, K., Mikami, A., Maeda, A,, Takahashi, H., Mishina, M., Haga, T., Haga, K., Ichiyama, A,, Kangawa, K. Kojima, M., Matsuo, H., Hirose, T. & Numa, S. (1986) Nature 323,411 -416. 64. Kubo, T., Maeda, A., Sugimoto, K., Akiba, I., Mikami, A,, Takahashi, H., Hagd, T., Haga, K., Ichiyama, A., Kangawa, K., Matsuo, H., Hirose, T. & Nama, S. (1986) FEBS Lett. 209,367- 372, 65. Peralta, E. G., Ashkenazi, A., Winslow, J. W., Smith, D. H., Ramachandran, J. & Capon, D. J. (1987) EMBU J . 6,39233929. 66. Libert, F., Parmentier, M., Lefort, A., Dinsart, C., Van Sande, J., Maenhaut, C., Simons, M. J., Dumont, J. E. & Vassart, G. (1989) Science 244, 569 - 572. 67. Parmentier, M., Libert, F., Maenhaut, C., Lefort, A,, Gerard, C., Perret, J., Van Sande, J. Dumont, J. E. & Vassart, G. (1989) Science 246, 1620-1622. 68. McFarland, K. C., Sprenghel, R., Philips, H. S., Kohler, M., Rosemblit, N., Nikolics, K., Segaloff, D. L. & Seeburg, P. H. (1989) Science 245,494-499. 69. Herskowitz, I. & Marsh, L. (1987) Cell50, 995-996. 70. Treuheart, J., Boeke, J. D. & Fink, G. R. (1987) Mol. Cell. Biol. 7,2316-2328. 71. Rose, M. D., Price, B. R. & Fink, G. R. (1986) Mol. Cell. Biol. 6,3490 - 3497. 72. Burkholder, A. C. & Hartwell, L. H. (1985) Nucleic Acid Res. 13,8463- 8475. 73. Nakayama, N., Miyanjima, A . & Arai, K. (1985) EMBO J . 4, 2643 - 2648. 74. Bouvier, M., Collins, S., O’Dowd, B. F., Campbell, P. T., De Blasi, A,, Kobilka, B. K., MacGregor, C., Irons, G. P. Caron, M. G. & Lefkowitz, R. J. (1989) J. Biol. Chem. 264,1678616792. 75. Sibley, D. R., Benovic, J. L., Caron, M. G. & Lefkowitz, R. J. (1987) Cell48,913-922. 76. Hausdorff, W. P., Bouvier, M., O’Dowd, B. F., Irons, G. P. Caron, M. G. & Lefkowitz, R. J. (1989) J. Biol. Chem. 264, 12657- 12665. 77. Sibley, D. R., Daniel, K., Strader, C. D. & Lefkowitz, R. J. (1987) Arch. Biochem. Biophys. 258,24-32. 78. Klein, C., Lubs-Haukeness, J. & Simons, S. (1985) J. Cell Biol. 100,715-720. 79. Klein, P., Knox, B., Borleis, J. & Devreotes, P. N. (1985) J . Biol. Chem. 260,1757-1764. 80. Vaughan, R. & Devreotes, P. N. (1988) J. Biol. Chem. 263, 14538- 14543. 81. Reneke, J. E., Blumer, K. J., Courchesne, W. E. & Thorner, J. (1988) Cell 55, 221 -234. 82. Konopka, J. B., Jenness, D. D. & Hartwell, L. H. (1988) Cell 54,609 - 620. 83. Bouvier, M., Hausdorff, W. P., De Blasi, A., O’Dowd, B. F., Kobilka, B. K., Caron, M. G. & Lefkowitz, R. J. (1988) Nature 333,370-373. 84. Van Haastert, P. J. M. (1987) J . Biol. Chem. 262, 7705-7710. 85. Bosch, L., Kraal, B. & Parmeggiani, A. (eds) (1989) Theguanine nucleotide binding proteins, Plenum, New York, London. 86. Neer, E. J. & Clapham D. E. (1988) Nature 333, 129- 134. 87. Freissmuth, M., Casey, P. J. & Gilman, A. G. (1989) FASEB J . 3, 2125-2131. 88. Lochrie, M. A. & Simon, M. I. (1988) Biochemistry 27, 49574965. 89. Birnbaumer, L. (1987) Trends Pharmacol. Sci. 8, 209-211. 90. Sullivan, K. A,, Miller, R. T., Masters, S. B., Beiderman, B., Heideman, W. & Bourne, H. R. (1987) Nature 330,758-760.

300 91. Masters, S. B., Stroud, R. M. & Bourne, H. R. (1986) Protein Engineering I , 47 - 54. 92. Strathmann, M., Wilkie, T. M. & Simon, M. I., (1989) Proc. Natl Acad. Sci. USA 86, 7407 - 7409. 93. Pupillo, M., Kumagai, A,, Pitt, G. S., Firtel, R. A. & Devreotes, P. N. (1989) Proc. Natl Acad. Sci. USA 86,4892-4896. 94. Kumagai, A,, Pupillo, M., Gundersen, R., Miake-Lye, R., Devreotes, P. N. & Firtel, R. A. (1989) Cell 57, 265-275. 95. Theibert, A. & Devreotes, P. (1986) J. Biol. Chem. 261, 15121 15125. 96. Van Haastert, P. J. M., Snaar-Jagalska, B. E. & Janssens, P. M. W. (1987) Eur. J . Biochem. 162,251 -258. 97. Van Haastert, P. J. M., De Vries, M. J., Penning, L. C., Roovers, E., Van der Kaay, J., Erneux, C. & Van Lookeren Campagne, M. M. (1989) Biochem. J . 258, 577-586. 98. Europe-Finner, G. N., Gammon, B., Wood, C. A. & Newell, P. C. (1989) J . CellSci. 93, 585-592. 99. Bominaar, A . A., Van der Kaay, J. & Van Haastert, P. J. M. (1991) Dev. Genet., in the press. 100. Coukell, M. B., Lappano, S. & Cameron, A. M. (1983) Dev. Genet. 3, 283 -297. 101. Kesbeke, F., Snaar-Jagalska, B. E. & Van Haastert, P. J. M. (1988) J . Cell B i d . 107, 521 - 528. 102. Snaar-Jagalska, B. E., Kesbeke, F., Pupillo, M. &Van Haastert, P. J. M. (1988) Biochem. Biophys. Res. Commun. 156, 757761. 103. Knecht, D. A. & Loomis, W. A. (1987) Science 236,1081 - 1086. 104. De Lozanne, A. & Spudich, J. (1987) Science 236,1086-1091. 305. Nakafuku, M., Obara, T., Kaibuchi, K., Miyajima, I., Miyajima, A,, Itoh, H., Nakamura, S., Arai, K. I., Matsumoto, K . & Kaziro, Y . (1988) Proc. Natl Acad. Sci. USA 85,1374- 1378. 106. Nakafuku, M.; Itoh, H., Nakamura, S. & Kaziro, Y. (1987) Proc. Nut1 Acud. Sci. USA 84, 2140-2144. 107. Dietzel, C. & Kurjan, J. (1987) Cell50, 1001- 1010. 108. Whiteway, M., Hougan, L., Dignard, D., Thomas, D. Y. Bell, L., Saari, G. C., Grant, F. J., O'Hara, P. & MacKay, V. L. (1989) Cell 56, 467-477. 109. Levitzki, A. (1988) Trends Biochem. Sci. 13, 298-301. 110. Nakayama, N., Kaziro, Y., Arai, K. & Matsumoto, K. (1988) Mol. Cell. Biol. 8, 3777 - 3783. 111. Jahng, K. Y., Ferguson, J. & Reed, S. 1. (1988) Mol. Cell. Biol. 8,2484 - 2493. 112. Cole, G. M., Stone, D. E. & Reed, S. I. (1990) Mol. Cell. Biol. Jn

1")