©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Family of Putative Receptor-Adenylate Cyclases from Leishmania donovani(*)

(Received for publication, April 7, 1995)

Marco A. Sanchez (1), David Zeoli (1), Elizabeth M. Klamo (2), Michael P. Kavanaugh (2), Scott M. Landfear (1)(§)

From the  (1)Department of Molecular Microbiology and Immunology and the (2)Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Leishmania parasites are exposed to pronounced changes in their environment during their life cycle as they migrate from the sandfly midgut to the insect proboscis and then into the phagolysosomes of the vertebrate macrophages. The developmental transformations that produce each life cycle stage of the parasite may be signaled in part by binding of environmental ligands to receptors which mediate transduction of extracellular signals. We have identified a family of five clustered genes in Leishmania donovani which may encode signal transduction receptors. The coding regions of two of these genes, designated rac-A and rac-B, have been sequenced and shown to code for proteins with an NH-terminal hydrophilic domain, an intervening putative transmembrane segment, and a COOH-terminal domain that has high sequence identity to the catalytic domain from adenylate cyclases in other eukaryotes. We have expressed the receptor-adenylate cyclase protein (RAC)-A protein in Xenopus oocytes and demonstrated that it functions as an adenylate cyclase. Although RAC-B exhibits no catalytic activity when expressed in oocytes, co-expression of RAC-A and RAC-B negatively regulates the adenylate cyclase activity of RAC-A, suggesting that these two proteins interact in the membrane. Furthermore, a truncated version of RAC-A functions as a dominant negative mutant that inhibits the catalytic activity of the wild type receptor. The rac-A and rac-B genes encode developmentally regulated mRNAs which are expressed in the insect stage but not in the mammalian host stage of the parasite life cycle.


INTRODUCTION

Parasitic protozoa of the genus Leishmania experience markedly different environments as they transit through their life cycle. Promastigotes live in the midgut of the sandfly vector, infectious metacyclic parasites reside in the mouthparts of the insect, and amastigotes live within the acidic phagolysosomal vesicles of the vertebrate host macrophages(1) . As the parasite migrates from the midgut to the mouthparts of the sandfly and subsequently into the phagolysosomes of the macrophage, it must be able to sense its changing extracellular milieu and respond by initiating a developmental transformation that culminates with the establishment of each successive life cycle stage. Although the factors that induce these developmental transformations are not fully understood, it seems likely that some environmental signals will act via binding of ligands to specific receptors in the parasite membrane which transduce the extracellular signal to the interior of the cell. However, to date no such ligands or their receptors have been identified in Leishmania parasites.

Nonetheless, a body of evidence suggests that the second messenger cAMP is probably involved in signal transduction events and in life cycle transformations in Leishmania and in other related kinetoplastid protozoa. Thus, various life cycle stages contain significantly different intracellular concentrations of cAMP in the related parasites Trypanosoma brucei(2) and in Trypanosoma cruzi(3) . Furthermore, cAMP analogs and inhibitors of cAMP phosphodiesterase promote the in vitro differentiation of non-infectious insect stage epimastigotes of T. cruzi into infectious metacyclic trypomastigotes(4) , whereas such reagents inhibit the transformation of amastigotes of Leishmania donovani into promastigotes (5, 6) or of long slender bloodstream forms of T. brucei into short stumpy forms that are infectious to the insect(7) . Perhaps the strongest case for the involvement of cAMP in the differentiation of kinetoplastid parasites is the observation that peptides which are digestion products of vertebrate -globin, and which are found in the gut of the triatomid insect vector following a blood meal, will activate a membrane-bound adenylate cyclase in non-infectious epimastigotes of T. cruzi and promote the differentiation of these epimastigotes into infectious metacyclic trypomastigotes in vitro(8) . These results underscore the importance for parasite development of environmental ligands which probably bind to receptors and activate adenylate cyclase.

Although the cognate ligands and receptors involved in such signaling processes have not been identified for any of these kinetoplastid protozoa, a family of genes encoding adenylate cyclases have been cloned in T. brucei(9) and in the related parasite T. equiperdum(10) . The proteins encoded by these genes possess a large extracellular hydrophilic NH-terminal domain which is separated by a putative transmembrane segment from a COOH-terminal domain that bears high sequence homology to the catalytic domains of adenylate cyclases from other eukaryotes. These trypanosome genes have been expressed in Saccharomyces cerevisiae where they complement a temperature-sensitive defect in the yeast adenylate cyclase(10, 11) . The structural organization of these trypanosome proteins suggests that they may be receptors whose NH-terminal domains bind specific extracellular ligands that activate the intracellular adenylate cyclase domain. This suggestion is reinforced by the structural similarity between these trypanosome proteins and several well-characterized receptors from higher eukaryotes(12) . Thus, mammalian atrial natriuretic peptide receptors bind their ligands via their NH-terminal extracellular domains and activate the cytoplasmic guanylate cyclase domain(13, 14) . However, ligands for these putative trypanosome receptors have not yet been identified, and their specific role in signal transduction is currently unknown.

The probable role of cAMP in life cycle transformations in kinetoplastid protozoa has led us to investigate adenylate cyclases in L.donovani. In this paper, we demonstrate that L. donovani parasites contain a family of genes that encode putative receptor-adenylate cyclases (RACs)()that are structurally very similar to the predicted proteins described in T. brucei and T. equiperdum. Five of these genes are arranged in a cluster in the L. donovani genome and are expressed as mRNAs of different sizes. One of these genes have been expressed in Xenopus oocytes and shown to encode a protein that functions as an adenylate cyclase. These results open the way to investigating the role of the Leishmania RAC proteins in signal transduction and their possible involvement in the life cycle transformations of this parasite. Studies on the RAC proteins of the intracellular Leishmania parasites may provide interesting parallels and distinctions compared to the RAC proteins identified in the extracellular parasites T. brucei and T. equiperdum.


EXPERIMENTAL PROCEDURES

Growth of Parasites and Isolation of Nucleic Acids

Promastigotes of either the DI700 (15) or the LV9 strain of L. donovani were cultured in Dulbecco's modified Eagle's-L medium (15) containing 5% heat-inactivated fetal calf serum (Life Technologies, Inc.) and 5% bovine embryonic fluid (Sigma) at 27 °C. For preparation of LV9 amastigotes, J774-G8 macrophage-like cells were cultured in plastic tissue culture flasks containing RPMI medium supplemented with 20 mM HEPES (Sigma), pH 7.3, and 20% heat-inactivated fetal calf serum at 37 °C in the presence of 5% CO and were infected with promastigotes at a multiplicity of 10 parasites/cell. After 24 h the medium and remaining promastigotes were aspirated, the adherent cells were washed twice with sterile phosphate-buffered saline, fresh medium was added to the culture, and the infection was continued for a further 24 h. Cells were removed from the tissue culture flask by scraping with a rubber policeman and were lysed by passage through a 30-gauge needle 20 times to achieve 95% breakage of cells, as monitored by staining with Trypan Blue. The liberated amastigotes were purified on a Percoll gradient as described(16) , and RNA was prepared immediately. Total parasite RNA was prepared by the guanidinium isothiocyanate-phenol method(17) .

Polymerase Chain Reaction

Degenerate oligonucleotides were purchased from Oligos Etc. and utilized in the PCR to amplify regions of the adenylate cyclase conserved catalytic domain from genomic DNA of L. donovani. The conserved peptide sequences and the oligonucleotides (bases in parentheses represent degenerate positions) designed from them were: peptide 1, TLIFTDI; oligonucleotide 1, AC(T/G/C)CT(T/C/G)AT(T/C)TT(T/C)AC(T/G/C)GA(T/C)AT; peptide 2, EVKTVGD; oligonucleotide 2, GA(G/A)GT(T/G/C)AAGAC(T/G/C)GT(T/G/C)GG(T/G/C)GA; peptide 3, GDSFMIA; oligonucleotide 3 (reverse complement), GC(A/G)ATCAT(A/G)AA(G/A/C)(G/C)(T/A)(A/G)TC(G/C/A)CC; peptide 4, RVGIHTG; oligonucleotide 4 (reverse complement): CC(G/A/C)GT(A/G)TG(A/G)AT(G/A/C)CC(G/A/C)AC(G/A/C)C(G/T). The degeneracies in these oligonucleotides were selected on the basis of the codon biases of previously sequenced Leishmania genes (18) . The PCRs were performed in a total volume of 50 µl and contained 10 mM Tris, pH 8.3, 50 mM KCl, 3 mM MgCl, 200 µM each dNTP, 2 µM each oligonucleotide, 2 µg of genomic DNA from the DI700 strain of L. donovani, and 1 unit of Taq Polymerase (Promega). Each PCR was performed on a Perkin Elmer DNA Thermal Cycler utilizing 30 cycles with a 95 °C melting temperature for 1 min, a 30 °C annealing temperature for 90 s, a 72 °C extension temperature for 1 min, with a final incubation at 72 °C for 5 min.

Isolation of Adenylate Cyclase Cosmid Clones

A library generated from a Sau 3A partial digest of genomic DNA from L. donovani strain DI700 and cloned into the cosmid vector SuperCos1 (Stratagene) was obtained from Dr. Buddy Ullman (Oregon Health Sciences University, Portland, OR) and screened with the 175-bp PCR product obtained using oligonucleotides 1 and 3. The product of this PCR was purified by phenol-chloroform extraction, and 150 ng was radiolabeled with [-[P]dATP (New England Nuclear) using random oligonucleotide priming(19) . Four nylon filters (Schleicher and Schuell) containing approximately 2,000 colonies each were screened by filter hybridization (20) uitlizing the radiolabeled PCR fragment at a concentration of 2 10 counts/min ml, and eight positive clones were colony purified for further analysis. Two of these positive cosmid clones, AC6 and AC9, which contained between them the entire rac gene family, were selected for detailed analysis.

DNA Sequencing

Segments of the AC6 and AC9 cosmids containing the coding regions of the rac-A and rac-B genes were subcloned into the plasmid vector Bluescript SK+ (Stratagene), and nested deletions were prepared by digestion with exonuclease III as described previously(21) . Single-stranded DNA was prepared from these deletions (22) and sequenced by the dideoxy chain terminating method(23) , as described (21) . In addition, some regions were sequenced utilizing synthetic oligonucleotides complementary to known regions of sequence. All sequences were obtained from both strands of the DNA. The partial sequences of RAC-C and RAC-D were obtained from double-stranded plasmid DNA using an A.L.F. automated DNA Sequencer (Pharmacia), and RAC-E partial sequence was obtained from double-stranded DNA by manual sequencing.

Adenylate Cyclase Probes

The AC-B probe was a 1.9-kb PstI restriction fragment from rac-B encoding amino acids 545-1174 (Fig. 1). This probe contains part of the conserved adenylate cyclase catalytic domain and part of the extracellular domain of RAC-B. The 1.4-kb AC-CD probe representing the conserved adenylate cyclase catalytic domain of RAC-A was prepared using the PCR and a forward primer (20-mer) initiating immediately downstream from the second putative transmembrane segment (II in Fig. 1) and a reverse primer initiating at the RAC-A stop codon and proceeding upstream for 20 nucleotides. The probes for the 3`-untranslated regions of rac-A and rac-B were a 1.0-kb EcoRI/SalI fragment and a 410-bp NheI/XhoI fragment, respectively, initiating 100 bp downstream from each stop codon. Hybridizations of Southern and Northern blots were performed in 50% formamide, 5 x SSC, 10 Denhart's solution, 0.1% SDS, 5 mM EDTA at 42 °C, and washes were performed in 0.1 SSC, 0.1% SDS, 5 mM EDTA at 55 °C.


Figure 1: Comparison of deduced amino acid sequences for RAC-A and RAC-B and alignment with T. brucei GRESAG4.3. Numbers at the right specify the last amino acid in each line. Vertical lines indicate identical amino acids. Predicted (42) transmembrane segments I and II are indicated by underlining. The alignments were generated using the program PILEUP(43) .



Expression of rac-A and rac-B mRNAs in Xenopus oocytes

For expression of rac mRNAs, the protein coding regions of rac-A and rac-B were subcloned into the oocyte expression vector pL2-5(24) . Capped transcripts were synthesized from these vectors utilizing T7 RNA polymerase as described previously(25) . Approximately 0.5 ng of each transcript, as well as 2.5 ng of transcript from a similar plasmid containing the coding region of the CFTR chloride channel, were injected into defolliculated stage V-VI oocytes from X. laevis as detailed previously(25) . After 4-5 days of incubation at 17 °C in ND-96 buffer containing 96 mM NaCl, 2 mM KCl, 1.8 mM CaCl, 1 mM MgCl, 5 mM HEPES, pH 7.4, membrane currents were measured from each oocyte using the two-microelectrode voltage clamp technique(26) .

Construction of a Dominant Negative Deletion Mutant of rac-A

For expression of a dominant negative version of the RAC-A protein, RAC-A, the plasmid containing the rac-A insert within the pL2-5 plasmid was digested with XhoI, religated, and transformed into bacteria. Plasmids prepared from individual colonies were tested by restriction digestion to identify those which were missing the terminal 0.7-kb XhoI fragment and hence were missing the sequence encoding the 218 COOH-terminal amino acids of RAC-A. This plasmid was transcribed with T7 RNA polymerase, and the RNA was microinjected into oocytes as described above.

cAMP Assays

Quantitation of cAMP was performed using the radioimmunoassay kit from Biomedical Technologies Inc. and the manufacturer's instructions. Oocytes were injected with 5 ng of either RAC-A or RAC-B cRNA and incubated in ND-96 for 3 days. Lysates were prepared from 15 oocytes containing each RNA and from 15 uninjected oocytes as follows. Each batch of oocytes was homogenized in 270 µl 10% trichloroacetic acid using a pestle and a microcentrifuge tube. This homogenate was clarified by centrifugation for 5 min in an Eppendorf microcentrifuge (Brinkmann Instruments, Inc.) for 5 min at 14,000 revolutions/min. The supernatant was extracted three times with 5 volumes of water-saturated ether, residual ether was removed by heating to 50 °C for 30 min, and the samples were stored at -20 °C. Aliquots of each lysate were diluted and assayed in duplicate by radioimmunoassay.


RESULTS

Cloning and Sequencing of Adenylate Cyclase Genes from Leishmania donovani

A PCR-based strategy was used to clone receptor-adenylate cyclase genes from L. donovani based on the assumption that they would contain sequences related to those found in the highly conserved catalytic domains of the trypanosome proteins (9, 10) . We chose four highly conserved peptides (see ``Experimental Procedures'') present in the trypanosome cyclases in order to design degenerate oligonucleotide primers (oligonucleotides 1-4). When these oligonucleotides were used in the PCR (27) in conjunction with genomic DNA from L. donovani, L. mexicana, or L. enriettii, they amplified DNA fragments of sizes 175 bp (oligonucleotides 1 and 3), 275 bp (oligonucleotides 2 and 4), and 400 bp (oligonucleotides 1 and 4) (data not shown). These are the sizes of fragments that would be predicted for PCR amplification from DNA sequences closely related to the catalytic adenylate cyclase domains of the trypanosome genes, and these results suggested that L. donovani does contain genes related to the trypanosome adenylate cyclases.

To determine the detailed structure of the Leishmania genes, we radiolabeled the 175-bp PCR fragment and used this reagent as a probe to screen a cosmid library of L. donovani genomic DNA. Two hybridizing cosmid clones, designated AC6 and AC9, were chosen for detailed structural analysis. The results of this analysis (see below and Fig. 3B) reveal that these two overlapping clones contain five clustered adenylate cyclase genes. Two of these genes, designated rac-A and rac-B, were then chosen for complete sequence analysis. The DNA sequence for each of these genes has been entered into the GenBank data base (rac-A and rac-B), and deduced protein sequences of rac-A and rac-B and their alignments with the T. brucei receptor-adenylate cyclase GRESAG4.3 (9) are shown in Fig. 1. The sequences of the RAC-A (1380 amino acids) and RAC-B (1331 amino acids) proteins clearly reveal that they are members of the receptor-adenylate cyclase family and are closely related in sequence and structure to the trypanosome proteins that are members of this family. RAC-A and RAC-B both contain: (i) a putative transmembrane segment near the NH terminus (underlined sequences marked I in Fig. 1), (ii) a large (approximately 830 amino acid) hydrophilic domain that follows this first transmembrane segment, (iii) a second putative transmembrane segment (underlined sequence marked II in Fig. 1), and (iv) a COOH-terminal hydrophilic domain with a high degree of identity to the conserved catalytic domain of eukaryotic adenylate cyclases. In addition to these structural similarities, there is a high degree of sequence identity between the trypanosome proteins and the RAC-A and RAC-B proteins. Direct comparison between each of the three proteins indicates that RAC-A is 30.6% identical to the T. brucei GRESAG4.3 receptor-adenylate cyclase, RAC-B is 31.2% identical to this trypanosome protein, and RAC-A is 55.5% identical to RAC-B. The highest degree of identity occurs over the conserved catalytic domains which are 52.7% identical between RAC-A and GRESAG4.3, 52.4% identical between RAC-B and GRESAG4.3, and 88.0% identical between RAC-A and RAC-B. Within the putative extracellular domains, the identities between the RAC-A, RAC-B, and GRESAG4.3 are clustered in islands of high identity separated by stretches of low identity.


Figure 3: Genomic arrangement of rac genes in L. donovani. A, Southern blot showing PstI digested DNA from L. donovani strain DI700 genomic DNA (genomic) or from cosmid clones AC9 and AC6. The blot was probed with the 1.9-kb PstI fragment of rac-B encoding part of the conserved adenylate cyclase catalytic domain (AC-B probe, ``Experimental Procedures''). Numbers (kilobase pairs) at the left indicate the mobility of DNA molecular weight markers. Letters A-E indicate the five hybridizing PstI fragments present in both genomic DNA and in the cosmid inserts. B, restriction map of the rac gene cluster indicating the exact (rac-A and rac-B) or approximate (rac-C, rac-D and rac-E) locations of the rac gene coding regions. The restriction map was generated from single and multiple digestions with different restriction enzymes (A, AseI; B, BglII; D, DraI; E, EcoRI; N, NotI; P, PstI; S, SpeI). Sites with asterisks denote a partial restriction map for this enzyme, indicating that other sites exist within the insert which have not been mapped. Hatched bars at the top of the figure indicate the positions of the inserts for the AC6 and AC9 cosmid clones. The open letters A-E underneath the restriction map define the location of the rac-A through rac-E genes. The positions of the coding regions of rac-A and rac-B (open boxes) are known precisely from the restriction maps and the DNA sequence of these coding regions. The arrows underneath these boxes indicate the direction of transcription for these genes. The position of the 2.7-kb PstI fragment containing the adenylate cyclase conserved catalytic domain of rac-E (black box) is known because a PstI restriction fragment of this size that hybridizes to an adenylate cyclase catalytic domain probe (AC-CD probe, ``Experimental Procedures'') is present at this location in the restriction map. Hence, this box defines the approximate position of the adenylate cyclase domain of rac-E. The 1.6-kb PstI fragment from rac-C is located between the EcoRI site and the right hand terminus of AC6; this region is shown as a cross-hatched box. The 1.2-kb PstI restriction fragment from rac-D that hybridizes to the adenylate cyclase probe is contained within the EcoRI/BglII restriction fragment of AC9, but is not present in AC6; hence, its approximate location is also shown as a cross-hatched box.



There are several other structural features that are worthy of comment. First, it is not clear whether the first putative transmembrane segment is retained in the mature proteins and tethers the NH terminus to the membrane or whether it is cleaved during maturation of the polypeptide, as is the case in the structurally related atrial natriuretic peptide receptor(14) . However, analysis of these sequences using von Heijne's algorithm (28) reveals a high similarity to known signal sequence cleavage sites at residue 53 of RAC-A and 59 of RAC-B, suggesting that these NH-terminal hydrophobic sequences may be removed during processing. Second, RAC-A and RAC-B both contain multiple consensus sequences for asparagine-linked glycosylation (29) (not shown), both in the putative extracellular domains and within the RAC-A conserved catalytic domain. However, the extent to which the RAC-A and RAC-B proteins are modified by glycosylation is currently unknown. Finally while the adenylate cyclase catalytic domains are highly conserved, the extreme COOH terminus of RAC-A (amino acids 1307-1380) and RAC-B (amino acids 1315-1331) bears no similarity to each other, suggesting that antibodies directed against these sequences should be able to specifically recognize each distinct isoform and may be useful for studies on subcellular localization of each protein.

Functional Expression of RAC-A and RAC-B Proteins in Xenopus oocytes

Although the rac-A and rac-B genes encode proteins with pronounced sequence similarity to known adenylate cyclases, it is nonetheless important to demonstrate experimentally that these Leishmania proteins possess adenylate cyclase activity. A strategy that was employed successfully with the adenylate cyclase genes from T. brucei and T. equiperdum was to complement a temperature sensitive adenylate cyclase mutant of S. cerevisiae by transformation with the trypanosome gene and demonstrate the ability of the recombinant yeast to grow at the non-permissive temperature. However, in similar experiments, we were unable to complement either temperature-sensitive or missense mutants of the yeast cyr1 adenylate cyclase gene; hence, we attempted expression of rac-A and rac-B in Xenopus oocytes. This novel approach involves co-expression of the rac gene with the human CFTR gene, which encodes a chloride channel that is stimulated by phosphorylation of its regulatory domain via the cAMP-activated protein kinase A(30) . Hence, if the rac genes encode functional adenylate cyclases, their co-expression in oocytes with CFTR will increase the intracellular cAMP, activate protein kinase A, stimulate the chloride channel activity of CFTR, and induce an increased current of Cl ions that can be measured by voltage clamping the oocytes. The results of one such experiment are shown in Fig. 2A. Oocytes that were injected with both rac-A and CFTR mRNAs (filled circles) exhibited increased transmembrane conductance compared to oocytes that were uninjected (crosses), injected with CFTR mRNA only (squares), or injected with rac-A mRNA only (open circles). This conductance increase exhibited a reversal potential of -25 to -30 mV, corresponding to the Cl equilibrium potential in Xenopus oocytes. This result strongly suggests that the RAC-A protein does possess adenylate cyclase activity which results in a CFTR-mediated Cl conductance increase. It is noteworthy that this adenylate cyclase activity is present in the absence of any exogenous ligands which might bind to the RAC-A extracellular domain and stimulate catalytic activity. A similar observation of enzymatic activity in the absence of added ligands was also reported for the trypanosome proteins expressed in yeast(10, 11) . Co-injection of several batches of oocytes with mRNAs encoding the rac-B protein and the CFTR protein produced no statistically significant increase in Cl conductances. These results suggest the possibility that there is no basal enzymatic activity of RAC-B in the absence of ligand or that it is significantly lower than that of RAC-A.


Figure 2: Adenylate cyclase activity of rac-A expressed in Xenopus oocytes. A, the cAMP-mediated activation of the CFTR Cl channel was measured by a two-electrode voltage clamp experiment. Oocytes were injected with RNA encoding CFTR alone (squares), RAC-A alone (open circles), RAC-A plus CFTR (filled circles), or uninjected controls (crosses). After incubation for 4-5 days, each oocyte was impaled with two electrodes and continually superfused with ND96 buffer. Each data point represents the steady state current (mean ± S.E., n = 3-4) at the indicated potential. Currents were recorded during a 250 ms pulse from a holding potential of -60 mV to the indicated test potential. B, the level of cAMP present in uninjected (nude) oocytes or in oocytes injected with 5 ng of cRNA encoding RAC-A (RAC-A), RAC-B (RAC-B), or RAC-A (RAC-A), or with cRNAs encoding both RAC-A and RAC-B (RAC-A + RAC-B), or RAC-A and RAC-A (RAC-A + RAC-A) was measured by radioimminoassay. The measurements were performed on lysates from 15 pooled oocytes of each type and represent the averages of two cAMP determinations. The largest difference between duplicates was in the RAC-A expressing oocytes, where the standard deviation was 135 pmol oocyte.



To confirm the results obtained by electrophysiology, we have assayed oocytes expressing RAC-A and RAC-B by a more conventional radioimmunoassay (Fig. 2B). This quantitation reveals that rac-A-injected oocytes accumulate significantly more cAMP than control uninjected oocytes but that cAMP levels are not elevated in rac-B injected oocytes. Furthermore, co-expression of RAC-A and RAC-B in the same oocytes led to a marked attenuation of the adenylate cyclase activity compared to the level observed in RAC-A expressing oocytes. To confirm that attenuation of RAC-A activity was not simply due to some contaminant in rac-B RNA which either degraded rac-A cRNA or inhibited its translation, we co-expressed rac-B RNA with cRNA encoding a Leishmania myo-inositol transporter. The co-expressing oocytes exhibited myo-inositol transport activity that was equivalent to that of oocytes injected with the transporter RNA only (data not shown); hence, the rac-B RNA does not contain an inhibitor that prevents expression of other co-injected RNAs. The inhibition or the RAC-A adenylate cyclase by RAC-B has been confirmed in three independent experiments and suggests that the RAC-B protein may interact with the RAC-A protein to negatively regulate its adenylate cyclase activity.

Expression of a Dominant Negative Form of RAC-A

Dominant negative forms of many signal transduction receptors can be prepared by expressing truncated proteins containing deletions in the cytoplasmic domain involved in transducing the signal(31) . Thus, deletion of part of the guanylate cyclase domain of the ANP receptor generates a truncated receptor that oligomerizes with the wild type protein and inhibits the guanylate cyclase activity of the complex(32) . We have prepared a truncated version of the rac-A gene that encodes a receptor, the RAC-A protein, that terminates at amino acid 1162 of the RAC-A protein and which is missing the last 218 amino acids of the adenylate cyclase catalytic domain. Co-expression of this truncated receptor with the wild type RAC-A protein (Fig. 2B, RAC-A + RAC-A) markedly inhibits the adenylate cyclase activity of RAC-A. In control experiments similar to those described above, the co-expression of rac-A RNA with inositol transporter RNA failed to inhibit functional expression of the transporter protein. Hence RAC-A functions as a dominant negative receptor, strongly suggesting that the wild type and deleted proteins physically interact in the oocyte membrane.

Genomic Arrangement of rac Genes

The existence of related but distinct rac-A and rac-B genes raises the question of how many members there are within this gene family. Since the catalytic domains of rac-A and rac-B are highly conserved, as is the case for adenylate cyclase genes in other eukaryotes(33) , a probe containing this domain should be able to detect other family members. Consequently, we utilized a 1.9-kb PstI fragment from rac-B (AC-B probe) containing part of this conserved domain to probe a Southern blot containing PstI digests of L. donovani genomic DNA and of AC6 and AC9 cosmid DNA. This blot (Fig. 3A) reveals five distinct bands of hybridization in genomic DNA marked A through E; bands A-D are also present in AC9 DNA, and bands B, C, and E are present in AC6 DNA. (In this nomenclature, the bands designated A and B are those derived from the rac-A and rac-B genes, respectively.) These results imply that the AC6 and AC9 inserts overlap, and that these cosmids contain between them all five hybridization bands present in the genomic DNA. This pattern suggests that the L. donovani genome contains at least five adenylate cyclase genes which are clustered together and hence present on two overlapping cosmid clones. Clustered arrays of functionally related genes are common among the kinetoplastid protozoa such as Leishmania and Trypanosoma and appear to comprise single transcription units which are synthesized as polycistronic precursor RNAs(34) .

To further investigate the arrangement of the multiple rac genes, we have constructed a restriction map of the AC6 and AC9 inserts, and we have mapped the locations of the adenylate cyclase genes by hybridization of restriction fragments to a second probe (AC-CD probe) derived exclusively from the conserved catalytic domain (Fig. 3B). This restriction map confirms that AC6 and AC9 do overlap and defines the precise location of rac-A and rac-B as well as the approximate locations of three other rac genes, designated rac-C, rac-D, and rac-E. Thus the open boxes in Fig. 3B represent the protein coding regions of rac-A and rac-B, as defined by the sequences of the subclones and the locations of specific restriction sites. These results also define the direction of transcription for these two genes (arrows under each gene) and the probable direction for transcription of the gene cluster. Together, these results suggest that there are five related rac genes located within this single cluster.

To confirm the existence of three additional rac genes, we subcloned the PstI fragments containing rac-C, rac-D, and rac-E and obtained partial DNA sequence from the termini of these subclones. The deduced amino acid sequences derived from one terminus of each subclone show pronounced similarity to the extracellular domains of RAC-A and RAC-B (Fig. 4A), whereas the deduced amino acid sequence derived from the other terminus of each clone shows identity to the adenylate cyclase catalytic domain of RAC-B (Fig. 4B). The similarities between each of the RAC proteins is shown schematically in Fig. 4C. These results confirm that each of these genomic fragments represents another rac gene and that there are five tandemly arrayed genes in this cluster. The limited sequence from RAC-E is identical to the corresponding regions of RAC-B, suggesting that these two proteins may be very closely related to each other; however, the presence of an additional PstI site in rac-B reveals that there is at least one polymorphism at the DNA level that generates the two differently sized rac-B and rac-E PstI fragments observed on the genomic Southern blot (Fig. 3A).


Figure 4: Partial deduced amino acid sequences derived from cloned PstI fragments encoding RAC-C, RAC-D, and RAC-E. Each subcloned PstI insert was partially sequenced from each terminus, and the deduced amino acid sequences were aligned with either RAC-A or RAC-B. In addition, an internal segment of the RAC-E insert was sequenced using an internal oligonucleotide primer. Amino acid identities are indicated by vertical lines. These sequences aligned with either the extracellular domain (RAC-C aligned maximally with RAC-A, whereas RAC-D and RAC-E aligned maximally with RAC-B) (A) or the catalytic adenylate cyclase domain of RAC-B (B). A schematic diagram (C) shows the regions of RAC-A and RAC-B that are homologous to the sequenced segments of RAC-C, RAC-D, and RAC-E. ECD refers to the extracellular domain, TMD to the transmembrane domain, and ACD to the adenylate cyclase catalytic domain.



Furthermore, the concordance between the five hybridizing PstI fragments present in genomic DNA (Fig. 3A) and the five hybridizing PstI fragments present in the AC6 and AC9 cosmid clones suggests that either: (i) this cluster contains all the adenylate cyclase genes present in the L. donovani genome, or that (ii) any other adenylate cyclase genes that might exist do not hybridize to the probe containing the conserved catalytic domain under the hybridization conditions used here and hence are relatively divergent in sequence. Another result which is consistent with a single clustered arrangement of adenylate cyclase genes is the observation (not shown) that Southern blots from pulsed-field gels (35) of L. donovani chromosomes reveal a single band of hybridization to the adenylate cyclase probe representing a chromosome of 850 kilobase pairs.

Expression of rac mRNAs during the Leishmania Life Cycle

To define the expression of the rac-A and rac-B mRNAs during the parasite life cycle, we have hybridized Northern blots containing equal amounts of total promastigote and amastigote RNA with probes representing the conserved adenylate cyclase catalytic domain and the unique 3`-untranslated regions of each mRNA (Fig. 5). These blots reveal that the rac-A (9 kb) and rac-B (5 kb) mRNAs are expressed in promastigotes but are not detectable in amastigotes; hence, these are developmentally regulated mRNAs. Rehybridization of these blots with a rRNA probe confirms that similar amounts of promastigote and amastigote RNA were present in each lane. The conserved adenylate cyclase catalytic probe hybridizes to bands of sizes 5, 7, and 9 kb, revealing that there are at least three different adenylate cyclase mRNAs. This probe also hybridizes to a 5 kb band present at a low level in the amastigote RNA, suggesting that one of the rac-C, -D, or -E mRNAs may be expressed in amastigotes and may comigrate with the rac-B mRNA.


Figure 5: Northern blots showing expression of rac mRNAs in promastigotes and amastigotes of L. donovani. Total RNA (10 µg) from promastigotes (P) and amastigotes (A) of the LV9 strain of L. donovani was separated on a 1.2% agarose-formaldehyde gel, blotted onto a nylon filter, and hybridized with probes representing the adenylate cyclase conserved catalytic domain (AC, hybridized with the AC-CD probe), or the 3`-untranslated regions of rac-A (rac-A) or rac-B (rac-B). To demonstrate that lanes contained similar amounts of total RNA, one of these blots was rehybridized with a rRNA (44) probe (rRNA). Numbers at the left indicate the mobility of RNA molecular weight markers indicated in kilobases.




DISCUSSION

The results presented here demonstrate that Leishmania parasites possess a family of putative receptor-adenylate cyclases that are related in structure to those described in the trypanosomes and that bear structural similarities to receptors with known ligands and functions from higher eukaryotes. Consequently, this family of probable receptors exists in kinetoplastid parasites that exhibit an intracellular mode of existence within their mammalian hosts and is not restricted to trypanosomes that live free in the bloodstream or to Kinetoplastida such as T. brucei and T. equiperdum that undergo antigenic variation as a mode of immune evasion. The two most significant questions that now require examination are: (i) what specific role do these putative receptors play in signal transduction, and (ii) what ligands do they recognize?

One possible role for the RAC proteins in signal transduction could be to bind ligands present in the insect vector or inside the host macrophages and induce a life cycle switch by elevating intracellular cAMP. The potential analogy to T. cruzi, where peptide ligands present inside the insect gut activate parasite adenylate cyclase and promote the developmental transformation between non-infectious epimastigotes and infectious metacyclics(8) , is obvious. It may be possible to assess the role of the RACs in development or in other physiological processes by genetic approaches, either by performing targeted gene disruptions (36) of individual rac genes, or by generating dominant negative mutants of these genes and overexpressing these altered polypeptides within the parasites. Finally, extensive studies on various RAC family members will be required to determine whether they all function in closely related signal transduction pathways and recognize different but possibly related ligands, or whether each RAC transduces a different signal with a distinct effect on parasite biochemistry and physiology.

The observation that co-expression of RAC-B markedly inhibits the adenylate cyclase activity of RAC-A (Fig. 2B) suggests an intriguing mechanism for receptor function. These two proteins may oligomerize in the membrane to maintain the innate adenylate cyclase activity of RAC-A in a repressed state. This repression might then be relieved by binding of ligand to the hetero-oligomer. If this interpretation is correct, it should be possible to detect complexes between RAC-A and RAC-B using specific antibodies against each protein or by epitope tagging individual receptors(32) . Furthermore, the observation that the truncated RAC-A protein functions as a dominant negative receptor (Fig. 2B) strongly suggests that these proteins oligomerize within the membrane and can affect the catalytic function of other members of the complex. The development of a dominant negative mutant of the rac-A gene also provides a genetic tool which may be used to illuminate the biological function of these receptors in the parasite.

Ligands for orphan receptor-guanylate cyclases have been identified in several higher eukaryotes(37, 38, 39, 40) , usually by assaying crude extracts for ability to stimulate guanylate cyclase activity in a target tissue followed by biochemical purification of the active peptide. By analogy, it may be possible to search for potential ligands for the RAC proteins by applying extracts of macrophages or of sandflies to intact parasites or to a heterologous system expressing individual rac genes and assaying for an increase in adenylate cyclase activity. The assay that we have used for detection of RAC-A adenylate cyclase activity involving activation of the co-expressed CFTR channel, or the more conventional radioimmunoassay, may be useful for identifying ligands for each putative receptor. Thus application of a mixture containing a ligand to oocytes expressing RAC-A or RAC-B or co-expressing both proteins may increase the cAMP level above that observed in the absence of ligand.

The observation that rac-A and rac-B mRNAs are detectable in promastigotes but not amastigotes implies that these putative receptors function either in the insect or during the transformation between the insect and the macrophage stage of the parasite life cycle. If these proteins are involved in developmental transitions, they could function in the switch between non-infectious procyclic and infectious metacyclic promastigotes(41) , or they could promote the differentiation of promastigotes to amastigotes by interacting with a ligand present in macrophages.

Clearly many questions remain to be answered concerning the functions of the rac genes and their protein products. At present, the RACs represent orphan receptors identified by molecular genetics but with unknown biological functions. However, as the first putative signal transduction receptors identified in Leishmania or related kinetoplastids(9, 10) , these proteins provide the opportunity to study signal transduction and its potential role in the life cycle of these medically important and biologically interesting parasites.


FOOTNOTES

*
This work was supported by Grant AI25920 and Research Career Development Award AI01162 (to S. M. L.), by Grant GM48709 (to M. P. K.) from the National Institutes of Health, and by a New Investigator Award in Molecular Parasitology (to S. M. L.) from the Burroughs Wellcome Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U17042 [GenBank® Link]and U17043[GenBank® Link].

§
To whom reprint requests should be addressed: Dept. of Molecular Microbiology and Immunology, Oregon Health Sciences University, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97201-3098. Tel.: 503-494-2426; Fax: 503-494-6862; E mail: landfear@ohsu.edu.

The abbreviations used are: RAC, receptor-adenylate cyclase protein; rac, receptor-adenylate cyclase gene or mRNA; PCR, polymerase chain reaction; bp, base pairs; CFTR, cystic fibrosis transmembrane conductance regulator; kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank Weibin Zhang for isolation and injection of Xenopus oocytes, Mark Drew for performing control experiments involving inositol transport assays on oocytes, and Richard Burchmore for culturing amastigotes of L. donovani. We also thank Buddy Ullman for thoughtful comments on this manuscript.


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