in Dictyostelium - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 86, pp. 4892-4896, July 1989

Biochemistry

Multiple a subunits of guanine nucleotide-binding proteins in Dictyostelium (signal transduction/receptor/chemotaxis)

M. PUPILLO*, A. KUMAGAIt, G. S. Pirr*, R. A. FIRTELt, AND P. N. DEVREOTES*1 *Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205-2185; and tDepartment of Biology, Center for Molecular Genetics, M-034, University of California, San Diego, La Jolla, CA 92093

Communicated by Alfred G. Gilman, March 31, 1989 (received for review February 14, 1989)

G protein a subunits.§ We also show that genes encoding these proteins are expressed at different times in development.

ABSTRACT Previous results have shown that chemotaxis and the expression of several classes of genes in Dictyostelium discoideum are regulated through a cell surface cAMP receptor interacting with guanine nucleotide-binding proteins (G proteins). We now describe cloning and sequencing of cDNAs encoding two Ga protein subunits from Dictyostelium. The derived amino acid sequences show that they are 45% identical to each other and to G. protein subunits from mammals and yeast. Both cDNAs are complementary to multiple mRNAs that are differentially expressed during development. This evidence and analysis of mutants presented elsewhere suggest that they have distinct physiological functions.

MATERIALS AND METHODS Oligonucleotide Screening. Oligonucleotides were 5' endlabeled using T4 polynucleotide kinase (Promega or New England Biolabs) and (y-32P]ATP (NEN or ICN). D. discoideum cDNA libraries were screened with two oligonucleotides at 50'C by the method of Wood et al. (12). The sequence of the first oligonucleotide was GGTAGCTCGGTAGAATCAGGTAAA. The sequence of the second oligonucleotide was

When deprived of nutrients, Dictyostelium discoideum amoebae cease growth and initiate a developmental program. Within a few hours cells, guided by chemotaxis and intercellular signaling, spontaneously aggregate and form a multicellular organism. Cells in specific positions of the multicellular structure differentiate into the stalk and spore cells of a mature fruiting body. The signal molecule is extracellular cAMP that interacts with a cell surface receptor. Present evidence indicates that the cAMP receptor also regulates aggregation stage and cell-type-specific gene expression, cell patterning in the migrating pseudoplasmodium, and morphogenesis during culmination (1-4). There appear to be two signal transduction pathways that may involve different kinetic classes of the cell surface receptor (5). The first is a "signaling" pathway in which cAMP, presumably binding to the rapidly dissociating class of receptors, results in the activation of adenylate cyclase with the subsequent synthesis and secretion of cAMP from the cell (6). Emitted cAMP can activate receptors on the same cell, creating a positive feedback loop, or on adjacent cells, relaying the signal. This response reversibly adapts and the cycle is repeated with a periodicity of 6-7 min (7, 8). A second, or "chemotactic," pathway is proposed to be mediated by the slowly dissociating class of cell surface receptors. This pathway is believed to involve activation of phospholipase C with the production of inositol 1,4,5triphosphate and diacylglycerol (9). Additional responses triggered by extracellular cAMP, such as activation of guanylate cyclase and the transient production of cGMP, actin polymerization, and myosin phosphorylation, also appear to be associated with the chemotactic pathway (10). Numerous biochemical studies indicate that both signal transduction pathways in Dictyostelium involve guanine nucleotide-binding proteins (G proteins). In addition, the primary structure of the cAMP receptor displays seven putative transmembrane domains, a structure identical to other G proteinlinked receptors (11). We now report the complete cDNA sequences and deduced amino acid sequences of two distinct

GGTGGTCAACAG

T

AGAACGTAAAAAAATGG.

Construction of Plasmids. cDNA inserts obtained from EcoRI digestion of phages were subcloned into the EcoRI site of pGEM2 or pSP73 (Promega) using standard techniques (13). RNA Blots. For the development time course of Gal and Ga2 expression, D. discoideum strain NC-4 cells were grown on SM (15 mM KH2PO4/5 mM K2HPO4/1% dextrose/1% bactopeptone/0.1% yeast extract/1% agar) plates in association with Klebsiella aerogenes, washed, and plated for development. RNA was isolated from cells harvested at specific times in development, sized on denaturing gels, and analyzed by RNA blot hybridization as described (1).

RESULTS Identification of Two Distinct a Subunits. cDNA clones for a subunits of G proteins were obtained by screening libraries prepared from 3.5-, 5-, and 11-hr stages of Dictyostelium development. Two oligonucleotide probes, corresponding to the highly conserved putative guanine nucleotide-binding (GGQRSERKKW) and GTPase (GAGESGK) regions of the known mammalian a subunit sequences were designed. These probes included 80-90o of the possible codons by biasing for the highly (A+T)-rich codon usage of Dictyostelium (14). An initial collection of 25 cDNAs was obtained from screens of about 250,000 plaques. These separated into two classes defined by the relative intensity of cross-hybridization and the presence or absence of an internal EcoPJ site (Fig. 1). Further restriction site analysis confirmed the existence of these two classes that were designated Gal (with an internal EcoRI site) and Ga2 (without an internal EcoRI site). The two Gal clones tested (RF1 and RF3) weakly cross-hybridized to the Ga2 class, whereas the Ga2 clone tested (FR1) did not crosshybridize to the Gal class. Two distinct size classes were Abbreviations: G protein, guanine nucleotide-binding protein; Go, G protein of unknown function; Gi, inhibitory G protein; G, stimulatory G protein; Tr, transducin. tTo whom reprint requests should be addressed. §The sequences reported in this paper have been deposited in the GenBank data base (accession nos. M25060 for Gal and M25061 for Ga2).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4892

Proc. Natl. Acad. Sci. USA 86 (1989)

Biochemistry: Pupillo et al. RF1-3' RF3-5' RF1 FR7

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4893

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found repeatedly among the Gal clones. RF1, FR7, FR11, AD1, AD3, and AD4 are less than or equal to 1.2 kilobases (kb), whereas RF3, RF5, and FR5 are 2.4 kb.

FIG. 1. Isolation of two sets of A cDNA clones. Several AgtlO and Agtll libraries were screened with the two oligonucleotides described in Materials and Methods. The first oligonucleotide was designated R in Baltimore and D in San Diego. The second oligonucleotide was designated F in Baltimore and A in San Diego. The clone designations indicate the oligonucleotides to which they hybridize. The plaque-purified A clones were spotted in grids onto lawns of Escherichia coli strain LE 392, lifted onto nitrocellulose, and probed with random primed cDNA inserts as indicated. In addition, subcloned inserts were digested with EcoRI Southern blotted, and probed with the same random primed inserts. *, Strong hybridization; *, weak hybridization; -, no hybridization above background. Hybridizations were carried out at high stringency.

The nucleotide sequence and the deduced amino acid sequence for Gal are shown in Fig. 2. Two clones, RF and AD4, were sequenced in both directions. The sequences

GM TTC CM AM MA AM CAC MT CAC MC TAC TAC MC MC I 43 AAC AAC MC MC CM AAA MT CM MA GTC AM ACA AMT TTA TM MT AAA ATA AM TM MT ATA TAT ATA CAT ATA MAA TAA ATA

130 ATG GGT MT ATT TGT GGT AM CCA GAA TTA GGA TCA CCA GM GAG ATT MA GCC MT CM CAT ATT MT AGT TTG TTG MA CM GCA 1 MET Gly Asn Ile Cys Gly Lys Pro Glu Leu Gly Ser Pro Glu Glu Ile Lys Ala Asn Gin His Ile Asn Ser Leu Leu Lys Gin Ala

217 AGA TCT AAA TTA GAG GGT GAA ATT AAA TTA TTA TTG TTA GGA GCA GGT GM TCA GGT AM TCA ACA ATC GCC AAA CAA ATG AAG ATT 30 Arg Ser Lys Leu Glu Gly Giu Ile Lys Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr Ile Ala Lys Gin MET Lys Ile 304 ATC CAT TTG AMT GGT TTC AAC GAT GAG GAG AAG TCA TCA TAT MA ACC ATC ATC TAC AAT AAT ACA GTT GGT TCA ATG CGT GTG TTG 59 Ile His Leu Asn Gly Phe Asn Asp Glu Glu Lys Ser Ser Tyr Lys Thr lie Ile Tyr Asn Asn Thr Val Gly Ser MET Arg Val Leu 391 GTA AAC GCC GCT GAA GM TTA MG ATT GGA ATC AGT GM MC MT MA GM GCC GCC TCT AGA ATC TCA MT GAT TTG GGC GAT CAT 88 Val Asn Ala Ala Glu Glu Leu Lys Ile Gly Ile Ser Glu Asn Asn Lys Glu Ala Ala Ser Arg Ile Ser Asn Asp Leu Gly Asp His

478 TTC MT GGT GTG TTG ACT GCA GAG TTG GCA CM GAT ATT AAA GCC CTT TGG GCA GAT CCA GGT ATT CM MT ACC TTC CAA AGA TCT 117 Phe Asn Gly Val Leu Thr Ala Glu Leu Ala Gln Asp Ile Lys Ala Leu Trp Ala Asp Pro Gly Ile Gin Asn Thr Phe Gin Arg Ser 565 TCA GAA TTC CAA CTA MT GAT TCA GCC GCT TAT TAC TTT GAT AGT ATC GAT AGA ATT AGT CM CCA TTA TAT TTA CCA TCT GMA AAT 146 Ser Glu Phe Gin Leu Asn Asp Ser Ala Ala Tyr Tyr Phe Asp Ser Ile Asp Arg Ile Ser Gin Pro Leu Tyr Leu Pro Ser Giu Asn

652 GAT GTT TTA CGT TCA AGA ACT MA ACA ACT GGT ATC ATT GAA ACA GTT TTT GAA ATT CAA MT AGT ACA TTT AGA ATG GTT GAT GTT 175 Asp Val Lau Arg Ser Arg Thr Lys Thr Thr Gly Ile Ile Glu Thr Vai Phe Giu Ile Gin Asn Ser Thr Phe Arg MET Vai Asp Vai 739 GGT GGT CAA AGA TCA GM AGA AAG MA TGG ATG CAT TGT TTC CAA GM GTT ACA GCA GTT ATC TTT TGT GTT GCC CTT AGT GAA TAT 204 Gly Gly Gin Arg Ser Giu Arg Lys Lys Trp MET His Cys Phe Gin Glu Vai Thr Ala Vat Ile Phe Cys Vai Ala Leu Ser Glu Tyr

826 GAT CTT AM CTT TAT GM GAT GAT ACT ACA AAT AGA ATG CM GAG TCA CTT MA CTC TTT AM GM ATA TGT MC ACC AM TGG TTT 233 Asp Leu Lys Leu Tyr Giu Asp Asp Thr Thr Asn Arg MET Gin Glu Ser Leu Lys Leu Phe Lys Glu Ile Cys Asn Thr Lys Trp Phe 913 GCA AAT ACT GCT ATG ATT CTT TTC TTA AAT MA AGA GAT ATT TTC TCT GAA MG ATT ACA MA ACA CCA ATT ACA GTT TGC TTC AAA 262 Ala Asn Thr Ala Met Ile Leu Phe Leu Asn Lys Arg Asp Ile Phe Ser Glu Lys Ile Thr Lys Thr Pro Ile Thr Val Cys Phe Lys 1000 GM TAT GAT GGT CCA CM ACT TAC GM GGT TGT TCA GAG TTT ATC AA CM CM TTT ATC AMT CM AAT GM MT CCA AAG A TCG 291 Giu Tyr Asp Gly Pro Gin Thr Tyr Glu Gly Cys Ser Glu Phe Ile Lys Gin Gin Phe Ile Asn Gin Asn Glu Asn Pro Lys Lys Ser 1087 ATC TAC CCA CAT TTA ACT TGT GCC ACT GAT ACT AMT MT ATC CTT GTT GTA TTC MT GCT GTC MA GAT ATT GTA TTA MT TTA ACT 320 Iie Tyr Pro His Leu Thr Cys Ala Thr Asp Thr Asn Asn Ile Leu Vai Vai Phe Asn Ala Val Lys Asp Ile Vai Leu Asn Leu Thr

1174 TTG GGT GAA GCT GGT ATG ATT CTT TM AGA ATC AA ACT TGA ACA CAC TAT TCA ATT TTT MA TM TAA TMA TM TM TM MA MA 349 Leu Gly Giu Ala Gly MET Ile Leu 1261 A

AGG AAT TC

FIG. 2. Nucleotide and deduced amino acid sequence of Gal. EcoRI inserts were subcloned into pSP73 or pGEM2 and sequenced directly with the dideoxynucleotide method using Sequenase.

4894

Biochemistry: Pupillo et al.

agreed completely with each other and with partial sequence of FR7 and RF3 (one of the larger class of clones). The longest open reading frame begins at the first AUG at nucleotide 130 and ends at nucleotide 1197. It encodes a polypeptide of 356 amino acids with a calculated molecular mass of 40,621 daltons. The internal EcoRI restriction site is at nucleotide 568. The nucleotide and deduced amino acid sequences for Ga2 are shown in Fig. 3. These were determined by overlapping clones D2 and AD6 and a polymerase chain reaction-generated copy of genomic DNA (15). The clones encompass 1174 base pairs (bp) and a complete coding sequence. The open reading frame begins at the first AUG at nucleotide 72 and ends at nucleotide 1142. It encodes a polypeptide of 357 amino acids with a calculated molecular mass of 41,323 daltons. Comparison of Primary Sequence of a Subunits. Fig. 4 shows an amino acid sequence comparison between the Gal and Ga2 subunits of Dictyostelium, yeast GPA1, mammalian G protein ofunknown function (GO), inhibitory G protein (GO), stimulatory G protein (Gs), and transducin (Tr). Gal and Ga2 are 45% identical to each other and 45% identical to mammalian G1, Go, and Tr. They are 35% identical to G. and GPA1 (18, 19). The predicted molecular masses are in the range of mammalian G proteins (39-45 kilodaltons) since the 107 amino acid "insert" found in GPA1 is not present in Dictyostelium a subunits. Five regions of the a subunits are highly identical. Four of these regions have been proposed as the sites for binding GTP I

Proc. Natl. Acad. Sci. USA 86 (1989)

based on analogy to c-Ha-ras and elongation factor Tu and are designated A, C, E, and G (19). The fifth region near the carboxyl terminus, which we designate region T, has not been previously described. Region A. This region is located at amino acids 36-52 in Gal and 31-47 in Ga2 and is proposed to be the site ofGTPase activity. A large portion of this region is 100% identical among all seven a subunits compared in Fig. 4. The sequence GAGESGK, from which one of the oligonucleotides was prepared, is included in the stretch of 100% identity. Region C. Region C is located at amino acids 201-207 in Gal and 203-209 in Ga2. With the exception of the initial position where F is replaced with V, Gal and Ga2 are identical to mammalian a subunits. GPA1 has two substitutions in this stretch. The stretch of highest identity among all seven a subunits includes the 3' half of this proposed site and 6 amino acids on the 3' side of the C site. This sequence, GGQRSERKKW, is the position of the second oligonucleotide. Region E. The E region includes amino acids 224-232 in Gal and 226-234 in Ga2. The differences between the a subunits in this stretch are clustered in five positions. By comparing the first four positions to Gal (amino acids 224, 226, 227, and 229), it is found that yeast differs in all four, Tr and G. differ in two positions, and Ga2 differs in one position. In the fifth position (amino acid 231 in Gal and 233 in Ga2), Gal, Ga2, and yeast are identical, whereas all of the mammalian a subunits are different.

GA ATT CCG ATT TAT TTA TM MC TAT ATA TAT ATA TAT MT TAT ATA AM TM AGA MA AM ACT TM AA

72 ATG GGT ATT TGT GCA TCA TCA ATG GM GGA GAA AM ACC MT ACT GAT ATT MT TTA TCT ATT GM MA GM AGA MA MG MA CAT 1 MET Gly lie Cys Ala Ser Ser Met Glu Gly Glu Lys Thr Asn Thr Asp lie Asn Leu Ser lie Glu Lys Giu Arg Lys Lys Lys His 159 MT GM GTT MA TTA TTA TTA CTT GGT GCT GGT GAA TCT GGT AM TCA ACA ATT TCA AM CM ATG MA ATT ATT CAT CM AGT GGT 30 Asn Glu Vai Lys Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr lie Ser Lys Gin Met Lys Ile lle His Gin Ser Gly

246 TAC AGT MT GM GM AGA AM GM TTT MA CCA ATT ATT ACA AGA MT ATT CTT GAT MT ATG AGA GTA TTA TTG GAT GGA ATG GGA 59 Tyr Ser Asn Glu Glu Arg Lys Glu Phe Lys Pro lie lie Thr Arg Asn lie Leu Asp Asn Met Arg Vai Leu Leu Asp Gly Met Gly 333 AGA CTT GGA ATG ACA )kTT GAC CCA AGT MT TCA GAC GCA GCA GTT ATG ATT MA GM TTA ACA TCA TTA CM GCA TCA ATT GTT ACA 88 Arg Leu Gly Met Thr lie Asp Pro Ser Asn Ser Asp Ala Ala Val Met lie Lys Glu Leu Thr Ser Leu Gin Ala Ser lie Vai Thr 420 GAT TGT TGG GGA GM TTA MT GM GAT CM GGT AA MG ATA AMA GCC TTA TGG ACA GAC CCA GGT GTC MA CAG GCA ATG AGA AGA 117 Asp Cys Trp Gly Glu Leu Asn Glu Asp Gin Gly Lys Lys Ile Lys Ala Leu Trp Thr Asp Pro Gly Val Lys Gin Ala Met Arg Arg

507 GCA AAT GAA TTT AGT ACA TTA CCA GAT TCA GCT CCA TAT TTC TTT GAT AGT ATA GAT CGT ATG ACA TCA CCA GTT TAT ATT CCA ACT 146 Ala Asn Glu Phe Ser Thr Leu Pro Asp Ser Ala Pro Tyr Phe Phe Asp Ser Ile Asp Arg Met Thr Ser Pro Val Tyr lie Pro Thr 594 GAT CM GAT ATT TTA CAT ACT CGT GTT ATG ACA AGA GGT GTT CAT GM ACA MC TTT GM ATT GGT MA ATC MA TTT AGA TTA GTA 175 Asp Gin Asp lie Leu His Thr Arg Val Met Thr Arg Gly Val His Glu Thr Asn Phe Glu lie Gly Lys lie Lys Phe Arg Leu Vai 681 GAT GTT GGT GGT CM CGT TCT GM AGA MG MA TGG TTA TCA TGT TTC GAT GAT GTT ACA GCA GTT GTA TTT TGT GTT GCC TTG TCC 204 Asp Vai Gly Gly Gin Arg Ser Glu Arg Lys Lys Trp Leu Ser Cys Phe Asp Asp Val Thr Ala Val Vai Phe Cys Vai Ala Leu Ser

768 GM TAT GAT TTA TTA TTG TAT GM GAT MT TCA ACC MT CGT ATG TTG GM AGT TTA CGT GTA TTC AGT GAT GTT TGC MT AGT TGG 233 Glu Tyr Asp Leu Leu Leu Tyr Glu Asp Asn Ser Thr Asn Arg Met Leu Glu Ser Leu Arg Vai Phe Ser Asp Val Cys Asn Ser Trp 855 TTT GTA MT ACT CCA ATC ATT TTA TTC TTA MC AA TCT GAT TTA TTC AGA GAG MA ATC MA CAT GTT GAT CTC TCT GM ACT TTC 262 Phe Vai Asn Thr Pro lle lie Leu Phe Leu Asn Lys Ser Asp Leu Phe Arg Glu Lys lie Lys His Val Asp Leu Ser Glu Thr Phe 942 CCA GM TAT MA GGT GGT AGA GAT TAC GM AGA GCC TCA MC TAT ATC MA GM CGT TTC TGG CM ATC MT MA ACC GM CM MA 291 Pro Giu Tyr Lys Gly Gly Arg Asp Tyr Glu Arg Ala Ser Asn Tyr lie Lys Glu Arg Phe Trp Gin lIe Asn Lys Thr Glu Gin Lys

1029 GCA ATC TAT TCT CAT ATC ACT TGT GCC ACC GAT ACA MT MT ATT CGT GTC GTT TTT GM GCT GTA MA GAT ATT ATT TTC ACT CM 320 Ala lie Tyr Ser His lie Thr Cys Ala Thr Asp Thr Asn Asn lie Arg Vai Val Phe Glu Ala Vai Lys Asp lie lie Phe Thr Gin 1116 TGT GTT ATG MA GCT GGT TTA TAT TCT TM MT MT TAT MT MT MA MA CGG MT TC 349 Cys Vat Met Lys Ala Gly Leu Tyr Ser

FIG. 3. Nucleotide sequence and deduced amino acid sequence of Ga2. EcoRI inserts were subcloned into pSP73 or pGEM2 and sequenced directly with the dideoxynucleotide method using Sequenase.

E L G S P GI: 1- 67 N G N I CG K P G G2: 1- 62 M G ICAS C I v Ss C T I G D E YE: 1-72 N G M A C T L S E E P GO: 1- 64 G AED GI: 1- 64 M G CT LS A AA EK TR: 1- 60 M G S C L GN S K T E D QR CS: 1- 80

MUG

GI:

68-129

G2:

63-130 YE: 73-245 GO: 65-128 GI: 65-127 TR: 61-123

GS: 81-150 A R

4895

Proc. Natl. Acad. Sci. USA 86 (1989)

Biochemistry: Pupillo et al.

N H I S L I K R S L E G K I L L L G A C E S G K S T I A K Q N K I I H| GN DF E E I K A N Q K I I NOS G Y S KR KHN E V K L L L L G A G E SG K S T IS K SIE TNTDINL C S G K S TV L K G GF N Q D P F L Q N K R A N D V I E O S L[EI Q R D K NE 1K LL L L G A G E S AA L E R S K A I E K L ED G I S A A K VK L L L L G A G E S G K S T IV K Q K I I H D G F S G S C K S T I V It Q K I I N E A C S E L L AA V E R S K I f R [1 LRJE D GYS L HSR E LK K L EDAEDAR T LLLLGES G K S T I V K Q K V G F G G GE E D P Q A K GE S G K S T IV T LLLL NE E K GQ R E AN K K D VV Y L

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FIG. 4. Comparison of deduced amino acid sequences with those of mammalian G proteins. Amino acid sequences for Gal (G1) and Ga2 (G2) were compared with those for yeast GPA1 (YE), bovine brain Go (GO), rat Gil (GI), bovine retinal transducin (TR), and rat G. (GS). Sequences were aligned with the program of Doolittle and collaborators (16, 17). Positions where either Gal or Ga2 is identical to at least two other G. proteins are boxed. Hatched bars below the sequence indicate the positions of the oligonucleotide sequences used in the initial screening. Capital letters A, C, E, G, and T indicate regions of the sequence discussed in the text.

Region G. The proposed G region contains residues 266278 in Gal and 267-279 in Ga2. Again the nonidentical residues are clustered in a few positions. In positions 273 and 277 of Gal, all of the a subunits differ from each other. In the remaining positions, Gal, Tr, and Gs have two substitutions and yeast has one. Region T. The fifth stretch of nearly complete identity is the sequence TCATDT near the carboxyl terminus of the polypeptides. This is located at positions 325-330 in Gal and 326-331 in Ga2. Only G, has one substitution in this stretch. Two areas in which the a subunits diverge significantly from each other are the amino-terminal 30-35 amino acids and the region between the A and the C sites. In Gal and Ga2 this second divergent area includes residues 62-197 and 57-199, respectively. This area can be subdivided into two regions with different degrees of identity. The 5' half (roughly amino acids 62-129 in Gal and 52-130 in Ga2) contains very few identities among the a subunits. However, the 3' half (amino acids 130-197 in Gal and 131-199 in Ga2) contains patches where several of the a subunits are identical. Both Gal and Ga2 contain the arginine (position 180 in Gal and 182 in Ga2), which, in mammalian Gs, is the site for ADP-ribosylation by cholera toxin (20). Neither a subunit has a cysteine as the fourth residue from the carboxyl terminus, which is the consensus site for ADP-ribosylation by pertussis toxin (21). Developmental Expression of Gal and G.2. RNA isolated from wild-type Dictyostelium cells (strain NC4) was probed

with Gal and Ga2 cDNA (Fig. 5). Gal hybridizes to multiple mRNAs. The predominant species of 1.7 kb is expressed at moderate levels in vegetative cells and increases to a maximal level at 10-12 hr, corresponding to the time ofloose aggregate formation in these experiments. Thereafter, RNA levels rapidly decrease. The 2.6-kb species has a similar pattern of regulation. The 2.2-kb RNA is preferentially expressed in multicellular aggregates. Ga2 also hybridizes to multiple RNA bands having different patterns of expression. The predominant 2.7-kb RNA is not expressed or expressed at very low levels in vegetative cells. Upon initiation of development, RNA levels increase, reaching a maximum level during aggregation and then declining. The 2.9-kb mRNA is present briefly very early in development. The two other mRNAs (1.9 kb and 2.3 kb) appear in sequence during the multicellular aggregate stage. Genomic Structure of a Subunits. Genomic mapping and isolation of genomic clones suggest that there is a single Gal gene and a single Ga2 gene (data not shown; ref. 22). This suggests that the multiple transcripts with dramatically different developmental time courses result from alternate splicing events or nested promoters.

DISCUSSION D. discoideum has at least two G protein a subunits, designated Gal and Ga,2. They are closely related to the mammalian G protein a subunits, being most similar in amino acid

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FIG. 5. Developmental regulation of Ga mRNAs. RNA samples isolated from wild-type NC-4 cells developed on filters and harvested at the times indicated were probed with Gal or Ga2 cDNA inserts. "O" represents RNA isolated from vegetative celis. In this developmental time course, visible aggregation initiated at -7-8 hr, streams were visible at -8-9 hr, loose aggregates were formed by 12 hr, and early culminants were found by 21 hr. Numbers on the right indicate the size of the RNAs in kb. sequence to Gai. Both subunits are potential substrates for ADP-ribosylation by cholera toxin. No physiological effects of cholera toxin on D. discoideum have been found, although ADP-ribose acceptors have been reported (23). Although pertussis toxin exerts its effects most frequently on Gi-like G proteins, such as Gal and Ga2, neither sequence contains the cysteine, which is the consensus site for ADP-ribosylation. However, biochemical effects of pertussis toxin on signaling in D. discoideum have been reported, suggesting that there may be at least one more Ga subunit (24). The previously unidentified T region (325-330) appears to be important since it is completely identical in all a subunits. The carboxyl terminus of a subunits has been implicated in receptor binding based on antibody blocking experiments (25). A recent report demonstrates that a peptide that encompassed this sequence blocks binding of Tr to rhodopsin (26). The most critical residue for the blocking effect was cysteine 321, which is the C of TCATDT. The A region is flanked by highly divergent areas (amino acids 3-35 and 60-125). The amino terminus of a subunits has been implicated in /3y subunits binding based on reconstitution assays with proteolytically cleaved Tr (27). At least two mammalian ,B subunits and two y subunits have been identified (28). Neer and Clapham (28) suggest that different combinations of , and 'y may be functionally different. If this is the case, the site of Py interaction with a may be expected to be a divergent area. The stretch 60-125 has been proposed to confer effector specificity. However, a chimeric a subunit containing the amino half of murine Gai2 and the carboxyl half of murine Gas stimulated adenylate cyclase (29). Either the

effector interaction site is not contained in this portion of the molecule or only part of the site is contained in this region. In addition, the stretch 60-125 corresponds to residues 73-245 in GPA1, which includes the 107 amino acid insert not found in Dictyostelium or mammalian a subunits. Both Gal and Ga2 probes are complementary to multiple, developmentally expressed RNAs, but DNA blot data suggest that both are encoded by single genes. At present, we do not know if the multiple mRNAs result from differential splicing or multiple transcription initiation sites. It is possible that some of the less abundant mRNA species complementary to these probes could be the products of other related Ga protein genes. Two lines of evidence strongly suggest that Gal and Ga2 have distinct functions. (i) Gal and Ga2 are as divergent from each other as they are from the a subunits of other organisms. (it) The mRNAs encoding these proteins have very different developmental patterns ofexpression. We have examined the regulation and function of Gal and Ga2 during growth and development (22). Our results indicate that the two proteins have distinct functions and may regulate different signal transduction systems. We thank R. Reed for helpful discussions and technical advice and S. Mann for supplying RNA samples. This work was supported by National Institutes of Health Grants to P.N.D. and R.A.F. and by a University of California Systemwide Biotechnology Grant to R.A.F. 1. Datta, S., Mann, S. K. O., Hjorth, A., Gomer, R., Howard, P., Armstrong, D., Reymond, C., Silan, C. & Firtel, R. A. (1987) in Genetic Regulation of Development (Liss, New York), pp. 33-61. 2. Devreotes, P., Fontana, D., Klein, P., Sherring, J. & Theibert, A. (1987) Methods Cell Biol. 28, 299-301. 3. Gerisch, G. (1987) Annu. Rev. Biochem. 56, 853-879. 4. Schaap, P. (1986) Differentiation 33, 1-16. 5. Janssens, P. M. W. & van Haastert, P. J. M. (1987) Microbiol. Rev. 51, 396-418. 6. Dinauer, M., MacKay, S. & Devreotes, P. (1980) J. Cell Biol. 86, 537-544. 7. Vaughan, R. & Devreotes, P. (1988) J. Biol. Chem. 263, 14538-14543. 8. Tomchik, K. J. & Devreotes, P. N. (1981) Science 212, 443-446. 9. Europe-Finner, G. N. & Newell, P. C. (1987) J. Cell Sci. 87, 221-229. 10. van Haastert, P. J. M. (1985) Biochim. Biophys. Acta 846, 324-333. 11. Klein, P., Sun, T. J., Saxe, C. L., Kimmel, A. R., Johnson, R. L. & Devreotes, P. N. (1988) Science 241, 1467-1472. 12. Wood, W. I., Gitochier, J., Lasky, L. A. & Lawn, R. H. (1985) Proc. Natl. Acad. Sci. USA 82, 1585-1588. 13. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 14. Kimmel, A. R. & Firtel, R. A. (1983) Nucleic Acids Res. 11, 541-552. 15. Engelke, D. R., Hoener, P. A. & Collins, F. S. (1988) Proc. Natl. Acad. Sci. USA 85, 544-548. 16. Feng, D. F., Johnson, M. S. & Doolittle, R. F. (1985) J. Mol. Evol. 21, 112-125. 17. Johnson, M. S. & Doolittle, F. (1986) J. Mol. Evol. 23, 267-278. 18. Nakafuku, M., Itoh, H., Nakamura, S. & Kaziro, Y. (1987) Proc. Natl. Acad. Sci. USA 84, 2140-2144. 19. Masters, S. B., Stroud, R. M. & Bourne, H. R. (1986) Protein Eng. 1, 47-54. 20. van Dop, C., Tsubokawa, M., Bourne, H. R. & Ramachandran, J. (1984) J. Biol. Chem. 259, 696-698. 21. West, R. E., Moss, J., Vaughan, M., Liu, T. & Liu, T.-Y. (1985) J. Biol. Chem. 260, 14428-14430. 22. Kumagai, A., Pupillo, M., Gundersen, R., Miake-Lye, R., Devreotes, P. N. & Firtel, R. A. (1989) Cell 57, 265-275. 23. Leichtling, B. H., Coffman, D. S., Yaeger, E. S., Rickenberg, H. V., al-Jumaliy, W. & Haley, B. E. (1981) Biochem. Biophys. Res. Commun. 102, 1187-1195. 24. Snaar-Jagalska, B. E., De Wit, R. J. W. & van Haastert, P. J. M. (1988) FEBS Lett. 232, 148-152. 25. Deretic, D. & Hamm, H. E. (1987) J. Biol. Chem. 262, 10839-10847. 26. Hamm, H. E., Deretic, D., Arendt, A., Hargrave, P. A., Koenig, B. & Hofmann, K. P. (1988) Science 241, 832-835. 27. Navon, S. E. & Fung, B. K. (1987) J. Biol. Chem. 262, 15746-15751. 28. Neer, E. J. & Clapham, D. E. (1988) Nature (London) 333, 129-134. 29. Masters, S. B., Sullivan, K. A., Miller, R. T., Beiderman, B., Lopez, N. G., Ramachandran, J. & Bourne, H. R. (1988) Science 241, 448-451.