Neuron,

Vol. 8, 631442,

April,

1992, Copyright

0 1992 by Cell Press

Identification of a Drosophila Gene Encoding a Calmodulin-Binding Protein with Homology to the trp Phototransduction Gene A. Marie Phillips, Ann Bull, and Leonard Department of Genetics University of Melbourne Parkville, Victoria Australia, 3052

E. Kelly

Summary We have isolated a number of Drosophila cDNAs on the basis of their encoding calmodulin-binding proteins. A full-length cDNA clone corresponding to one of these genes has been cloned and sequenced. Conservation of amino acid sequence and tissue-specific expression are observed between this gene and the transient receptor potential (frp) gene. We propose the name transient recepfor potential-like (frpl) to describe this newly isolated gene. The frpl protein contains two possible calmodulin-binding sites, six transmembrane regions, and a sequence homologous to an ankyrin-like repeat. Structurally, the frpl and frp proteins resemble cation channel proteins, particularly the brain isoform of the voltage-sensitive Ca2+ channel. The identification of a protein similar to the frp gene product, yet also able to bind Ca*+/calmodulin, allows for a reinterpretation of the phenotype of the frp mutation and suggests that both genes may encode light-sensitive ion channels. Introduction The Ca*+-binding protein calmodulin, originally identified as a regulator of cyclic nucleotide phosphodiesterase (Cheung, 1971), can act, in the presence of micromolar intracellular concentrations of free Ca2+, to regulate a number of cellular processes. Although originally isolated from mammalian tissues, calmodulin is highly conserved throughout the eukaryotic phyla. Calmodulin has been purified from Drosophila and shown to activate mammalian cyclic nucleotide phosphodiesterase (Yamanaka and Kelly, 1981). More recently, the Drosophila calmodulin gene has been cloned and sequenced (Yamanaka et al., 1987). The calmodulin protein has the capacity to bind 4 mol of Ca2+ per mol of protein and contains four Ca*+-binding domains described as “EF hands” (Kretsinger, 1980). It is known to exist in several different conformations according to the number of bound Caz+ ions (Klee, 1977; Dedman et al., 1977; Wolff et al., 1977; Seamon, 1980), with different target enzymes recognizing and binding the different conformers. These differences in conformation allow calmodulin to translate quantitative alterations in Ca*+ concentrations into a qualitative biological response. Evidence for such a regulatory mechanism has been obtained from the study of the interaction between Caz+/calmodulin and enzymes involved in the synthesis and degradation of cyclic nucleotides. In mamma-

lian brain, both adenylate cyclase (Brostrom et al., 1975; Cheung et al., 1975) and CAMP phosphodiesterase (Cheung 1971) can be regulated by Ca*+/calmodulin. At low Ca*+ concentrations, Ca*+/calmodulin activates adenylate cyclase to produce CAMP, with half-maximal stimulation at 0.08 PM Ca*+. As Ca*+ levels rise, the adenylate cyclase is inhibited (halfmaximal inhibition at0.3 pM)and theCa*+/calmodulin complex increases its affinity for and activates the phosphodiesterase (half-maximal stimulation at 0.3 PM) (Piascik et al., 1980). Thus these two enzymes appearto be regulated inverselywithin the physiological range of intracellular Ca*+ concentration. In Drosophila Ca*+lcalmodulin-dependent adenylate cyclase (Ross and Gilman, 1980) and cyclic nucleotide phosphodiesterase (Yamanaka and Kelly, 1981; Kauvar, 1982) have been described, and a Drosophila learning mutant, rutabaga, has been shown to be defective in the cyclase (Aceves-Pina et al., 1983; Livingstone et al., 1984; Dudai and Zvi, 1984). This suggests that the interaction between the intracellular levels of free Ca2+ and the production of CAMP may be important in the learning process. Calmodulin hasalso been implicated in neural function in mammals via its activation of Ca*+/calmodulindependent protein kinases (Kennedy et al., 1983; Nairn and Greengard, 1987). Activation of these enzymes has been shown to modulate neurotransmitter release (DeLorenzo et al., 1979; Llin et al., 1985), and it has been proposed that this enzyme might be central to the mechanism of short-term memory (for review see Teyler and Di Scenna, 1987). A Ca*+/calmoduIindependent phosphoprotein phosphatase has been isolated from mammalian tissues and shown to be identical to the calmodulin-binding protein calcineurin (Stewart et al., 1983). In Drosophila, Ca2’/calmodulin-dependent protein kinase activity has been identified (Kelly, 1983; Leonard and Kennedy, 1988, Sot. Neurosci., abstract), and more recently, the Drosophila gene encoding the type II isoform of Ca*+/ calmodulin-dependent protein kinase has been cloned and sequenced (Cho et al., 1991). A phosphoprotein phosphatase activity analogous to calcineurin has been identified in Drosophila (Orgad et al., 1987), but as yet no mutations in either the calcineurin or the Caz+/calmodulin-dependent protein kinase gene have been reported. Many more proteins, in addition to those described todate, presumably respond toelevation in intracellular Ca*+ via calmodulin. The investigation of calmoduIin-regulated proteins is madedifficult bytheabsence of a specific amino acid sequence motif that identifies the binding site for Ca2’lcalmodulin. Instead it appears that Ca*+/calmodulin recognizes and binds to an amphiphilic a helix in the target protein (Buschmeier et al., 1987; O’Neil and DeGrado, 1990). Oneway in which these proteinscan be investigated is by using

Neuron 632

12345678

ABCD

4 97kd 4 66kd

431kd

Figure 1. Identification ern Blots of Drosophila modulin as a Probe

of Calmodulin-Binding Proteinson WestHead Fractions Using 1*51-Labeled Cal-

Lanes A and Care crude membrane fractions tions from heads of Oregon-R wild-type flies, B and D are similar fractions from white-eyed

and cytosolic fracrespectively; lanes mutant flies.

calmodulin as a ligand to identify and clone cDNAs for calmodulin-binding proteins (Sikela and Hahn, 1987). Sequencing the resulting cDNAs may help identify novel calmodulin-regulated proteins. We have initiated a molecular genetic analysis of calmodulinregulated proteins present in the Drosophila nervous system. Using radioiodinated calmodulin as a probe on a cDNA library in the expression vector hgtll, we have identified three Drosophila genes that encode calmodulin-binding proteins. A full-length cDNA clone corresponding to one of these genes has been analyzed and found to encode a novel protein that may play a role in the phototransduction process. Here we discuss the sequencing and characterization of this clone. Results Radioiodinated Calmodulin Recognizes Drosophila Proteins on Western Blots Western blots of both cytosolic and crude membrane fractions of Drosophila heads (see Experimental Procedures) were prepared and probed with 1251-labeled calmodulin. A number of calmodulin-binding proteins were identified (Figure 1). In total, some 14 different protein bands, ranging in molecular weight from 34,000 to 200,000, were found to bind radioiodinated calmodulin. The subsequent washing of these Western blots in buffer containing 1 mM EGTA resulted in a considerable reduction of the radioactivity associated with all of the bands (data not shown), indicating that these proteins bind calmodulin in a Ca*+-dependent manner. Interestingly, low molecular weight calmodulin-binding proteins (30,000-45,000) are found only in

Figure 2. A Western Blot of Proteins from Carrying lgtll Putative Calmodulin-Binding Probed with rz51-Labeled Calmodulin

Lysogenic Protein

Bacteria Clones,

Lane 1, Sgtll without an insert (control); lane 2, clone AB3.7; lane 3, clone AB3.9; lane 4, clone AB2.17; lane 5, clone AB3.33; lane 6, clone AB3.2; lane 7, clone AB3.13; lane 8, clone AB3.14. Inter se hybridization studies indicate that clones AB3.9, AB3.33, AB3.2, and AB3.14 derived from the same transcript, clones AB 2.17 and AB3.13 derive from a second transcript, and clone AB3.7 from a third.

the cytosolic fraction, whereas high molecular weight proteins (>96,000) arefound mainly in the crude membrane fractions. Screening an Expression Library for Clones Encoding Calmodulin-Binding Proteins We have used radioiodinated calmodulin as a ligand probe to identify clones encoding calmodulin-binding proteins(Sikelaand Hahn, 1987) from a Drosophila head hgtll cDNA expression library (Itoh et al., 1985). In a screen of 106 plaques, some 50 clones were originally designated as being positive for calmodulin binding. Of these clones, 24were purified to homogeneity using the same screening procedure and were subjected to further analysis. Lysogens were prepared from I5 of the clones and induced, and the lysates run on polyacrylamidegels. Western blots from thesegels were then probed with radioiodinated calmodulin. A selection of clones that were probed in this way, along with a control lysate produced from a lysogen made from hgtll without an insert, is shown in Figure 2. With the exception of thecontrol, which produced no calmodulin-binding proteins, all of the clones tested positive for oneor morecalmodulin-binding polypeptides. Some of the clones produced a single fusion protein (e.g., clones AB 3.7 and AB2.17), whereas others appeared to be proteolysed, giving either two high molecular weight bands (clone AB3.14) or much smaller bands (clone AB3.9). The Xgtll isolates were subcloned into pUC19, and the inserts from the sub-

trpl: A Novel 633

Drosophila

Calmodulin-Binding

Protein

A 6

the introduction of a stop codon and the truncation of the open reading frame in AB3.14, compared with AB3.14IZ9 (see Figure 4A). The sequence of the AB3.9 clone is identical to that of AB3.14/Z9 in this region. As the original X.AB3.14 lysogen produced a fusion protein considerably larger (160 kd) than that expected if the stop codon were present (130 kd), this base pair change is likely to be an artifact of subcloning.

4kb.N’

Figure 3. Northern Blot of Total RNA from of Drosophila Probed with pAB3.14 Lane A, RNA from mens.

clones

were

heads;

used

lane B, RNA from

to

probe

the Oregon-R thoraxes

Southern

Strain

and

blots

abdo-

of

the

EcoRI-digested h clones. The 24 clones were found to fall into one of three cross-hybridizing groups, indicating that we have so far identified genes for at most three transcripts that may encode polypeptides shown in Figure 1. The remainder of this report deals with the characterization of one these transcripts.

The AB 3.14 Transcript Adult Head

Is Highly

Expressed in the

The clones AB3.14, containing a 1.7 kb insert, and AB3.9, containing a 1.1 kb insert,werechosen as being representative of one of the three transcription units. A Northern blot of total RNA prepared from heads probed with the AB3.14 insert, showed only a single species of RNA with an approximate size of 4.0 kb, whereas no signal was detected in whole RNA prepared from thoraxes and abdomens even after prolonged exposure (Figure 3).

Isolation and Sequencing of a Full-Length Clone Corresponding to AB3.14

cDNA

To isolate a full-length cDNA, the AB3.14 insert was used to probe a second Drosophila head cDNA library in the hZAP vector. From this screen, a clone, pAB3.141 Z9, with an insert size of 3.7 kb was identified as a potential full-length cDNA. The restriction enzyme map of the 3’ end of this second clone was shown to be identical with that of the AB3.14 insert. The AB3.14, AB3.9,andtheAB3.14/29cDNAswerethensequenced (Sanger et al., 1977), and the amino acid sequences of these clones were deduced. The AB 3.14129 clone has a single open reading frame immediately preceded by a Drosophila consensus initiation sequence (Cavener, 1987) and is expected to produce a polypeptide of 127.6 kd. Two base pair changes were observed between theAB3.14and AB3.141Z9 clones. One base pair change would have no effect on the amino acid sequence, but the second, a G to T transversion, causes

The complete amino acid sequence derived from AB3.14lZ9was compared with those in the data banks, in the hope of finding homology with other known calmodulin-binding proteins. Surprisingly, the greatest homology was found with the Drosophila phototransduction gene transient receptor potential (trp) (Monte11 and Rubin, 1989; Wong et al., 1989), with an overall amino acid identity of 39%. The amino acid sequences encoded by AB3.14lZ9 and the trp gene are compared in Figure 4A. Because of the obvious homology between the proteins encoded by AB3.14lZ9 and trp, we propose that this new gene be called transient receptor potential-like (trplh The amino acid identity between the two proteins is not uniformly distributed throughout the sequences. There is considerable (56%) amino acid identity over the aminoterminal amino acids (residues 50-340). The central region consists of two segments: the amino-terminal segment (residues 340-500), with 29% identity, and the remainder (residues SOO-700), which shows 74% identity. The level of identity falls off dramatically (to 17%) over the carboxy-terminal regions of the proteins.

Homology of the Transmembrane trpl and Channel Proteins

Regions of the

As well as the homology with the vey of the data bases also showed

trp protein, that the trpl

the surprotein

is homologous, in part, to various channel proteins. Like trp, the trpl protein contains a hydrophobic central region (Figure 48). Using an algorithm to predict membrane-spanning regions (Klein et al., 1985), five possible transmembrane segments can be identified, (Sl, S2, S4, S5, and S6 in Figure 48). Comparison of these sequences with those in the data bases revealed homology with both Caz+ and Na’ channel proteins. In fact, thesecomparisons suggested that asixth transmembrane region (S3) may exist in the trpl protein. Figure 5 shows a sequence comparison between the putative S3 region in the trpl protein and the S3 regions of each of the four homologous domains from the a, subunit of the brain dihydropyridine-sensitive voltage-dependent Ca*+ channel (Hui et al., 1991). The conservation of the asparagine, aspartate, and leucine residues in the trpl protein and all but one of the Cal+ channel S3 regions, along with similarity in the amino acid sequence immediately preceding the S3 region, suggests that this may indeed be a membranespanning region that is not detected by the algorithm. Having observed this homology between the voltagedependent Ca*+ channel and trpl proteins, further

Neuron 634

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comparisons were made between the presumptive membrane-spanning regions of the two polypeptides. The S6 region of the trpl protein also shows homology with the S6 regions of the Ca2+ channel protein (Figure 5). Most noticeable here is the conservation of the phenylalanine and aparagine residues in all of the S6 regions. The S4 regions of all voltage-gated cation channels are thought to act as voltage sensors (Noda et al., 1984). Some homology is observed between the S4 regions of the trpl and the Ca2+ channel proteins (Figure 5), but most importantly, in the series of positively charged amino acids every third residue is absent from the trpl S4 region. However, with a single exception, these positively charged residues in the Ca2+ channel S4 regions are replaced by polar amino acids in the trpl protein. Even in the absence of these positively charged residues, there is still considerable homology between the trpland Ca*+ channel S4 regions. Comparison of the presumed S5 sequence of the trpl protein with the S5 regions of the Ca2+ channel protein (Figure 5) shows that the sequence of the trpl S5 region looks like a fusion of the Ca2+ channel S5 regions from the first and fourth homology domains. The first two membrane-spanning regions, Sl and S2, are the least conserved within and between channel proteins, and little or no homology can be detected between these regions of the trpl protein and the equivalent regions of the Ca2+ channel protein. In fact, these two sequences also represent regions of low homology between the trp and trpl proteins. It has been suggested that a conserved region between S5and S6 may also partially span the membrane and act as the extracellular entrance to the transmembrane pore. In thevoltage-sensitivechannels, the negatively charged residues in this region are thought to neutralize some of the positive charge on the S4 region (Guy and Seetharamulu, 1986, Greenblatt et al., 1985). The regions, known as SSI and SS2, from both the Ca* channel and the trpl proteins are also compared in Figure 5. Although there is some homology

I

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Amino

Acid

Sequence

and

Hydrophobicity

Profile

(A) Predicted amino acid sequence of the protein encoded by the AB 3.14/29 (trpf) cDNA and comparison with the amino acid sequence of the trp protein, determined by Monte11 and Rubin (1989). The central putative transmembrane regions of the trpl protein are underlined and labeled Sl-S6 (see [B]). The dotted underlined regions in both proteins are the single copies of the ankyrin-like repeat motif.Thedouble-underlined regionsare the presumptive calmodulin-binding sites in the trpl protein. The arrow labeled “a” indicates the presence of possible CAMPdependent protein kinase phosphorylation sites in the trpl protein. The positions in the polypeptide chain where the AB3.14 and AB3.9cDNAs initiate are indicated bythetwo labeled arrows. The glycine residue at position 843 that precedes the premature stop codon in AB3.14 is underlined. (B) Hydrophobicity plot of the trpl protein. The presumptive membrane-spanning regions are shown as Sl-S6. The 53 region is defined by homology with the a, subunit of the brain Ca2+ channel (see Figure 5).

trpl: A

Novel

Drosophila

Calmodulin-Binding

Protein

635

Figure5. Comparison of the Various Membrane-Spanning Segments of the a, Subunit of the Brain Ca*+ Channel with the Equivalent Regions of the frpl Protein

MV~D~KFFIYTLVL~A~-GLNQLL VALIAMLFFIYAV-------IGM-QMF LLLLLFLqL------L[M-5F ALLVLFVIIIYAI-------IGM-ELF M~----TTLLQBM~IGM-

IV

trp1 Ca Ca Ca Ca

I II III I"

Channel Channel Channel Channel

identical amino acids are boxed. The asterisks below the amino acid residues in the S4 region indicate the regular pattern of basic amino acids found in voltage-sensitive channels. All but one of these residues are absent in the trpl protein.

;;:;;;;!;:I

q

m

Ca Channel

LF

56 homolouy

SSl

trp1 Ca Ca Ca Ca

channel channel channel channel

I II III IV

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channel channel channel channel

I II III IV

552

between the trpl and Ca*+ channel proteins in this region, it is not extensive. Homologies similar to, though less extensive than, those shown above are also observed between trpl and the rat Na+ channel transmembrane domains (Noda et al., 1986) (data not shown). Identification of Possible Calmodulin-Binding Sites in the trp/ Protein Calmodulin is known to bind toaminoacid sequences that can form amphiphilic a helices with one face of the helix positively charged (Buschmeier et al., 1987; O’Neil and DeGrado, 1990). Two regions that show a high probability of forming an amphiphilic a helix were found in the trpCencoded protein (residues 710727 and 809-825 inclusive in Figure 4A). When drawn in the form of an a helical wheel (Schiffer and Edmundson, 1967), these sequences produce amphiphilic helices with a positively charged face and a hydrophobic face, as expected of calmodulin-binding sites (Figure 6A). Both of these sequences are contained within the polypeptide encoded by the AB3.14 clone, whereas the second putative site is present only in the AB3.9 clone. The sequence spanning residues 809-825 is the only candidate for the calmodulin-

binding site common to both clones. Whether residues 710-727 represent a second calmodulin-binding site is not clear. It more closely resembles the consensus motif, also containing the conserved tryptophan residue and a serine residue that could act as a substrate for CAMP-dependent protein kinase; the latter is a featurecommon to manycalmodulin-binding peptides (Buschmeier et al., 1987). A region homologous to the first putative calmodulin-binding site in the trpl protein is not present in the trp protein; however, the second site does show considerable homology (47% identity) with the equivalent region in the trp protein. The trp/ and frp Proteins Both Contain an Ankyrin-like Repeat Domain Residues 153-182 of the trpl protein and the equivalent region in the trp protein (Figure4) are of potential significance. These sequences from the trpl and trp proteins are comparable to the “ankrepeat” consensus sequence (Lux et al., 1990) (Figure 6B). Both the trp and the trpl sequences conform well to the consensus sequence, and in fact, both sequences show considerable identity with individual members of the ankrepeats both from ankyrin (Lux et al., 1990) and from the Drosophila Notch protein (Wharton et al., 1985).

Neuron 636

Z9 clone, hybridized only to the the eye in cryostatic sections of indicating the presence of trpl Hence we can say that both trpl in the same tissue, although at delineate the expression of trpl tinula cells.

hydrophobic

hydrophilic

retinula cell layer of adult flies (Figure 7B), mRNA in this tissue. and trp are expressed this stage, we cannot exclusively to the re-

Discussion

R

D

SVKWVIRIFRKSSKTIDR

hydrophilic

R

R

RRIKVWERRLMKGFQVAP B *** ** ** ** * -G-TPL”-AA-------“--LL--GA------DPDITPLMLAAWKNNFEILRILLDRG~VPVPHDI VDITPLILAAHRNNYEILKILLDRGATLPVPHDV Figure 6. Putative peat-like Domains

Calmodulin-Binding of trppl

ANKREPE,AT consensus TRPI. TRP

Sites

and

Ankyrin

Re-

(A) Helical wheel diagrams of the putative calmodulin-binding regions encoded by &p/amino acids 710-727 and 809-825 showing the presence of a basic hydrophilic face and a hydrophobic face. The serine that is a potential CAMP-dependent phosphorylation site in the first helix is shown as a boxed “P”. (B) Comparison of the amino acid sequence of residues 153-182 in the trpl protein and the equivalent region of the trp protein with the consensus sequence derived from the various ankyrin repeat domains (Lux et al., 1990).

Genetic localization Expression of trp/

and Tissue-Specific

The genomic location of the trplgene was determined by in situ hybridization of trp/cDNA clones to salivary gland polytene chromosomes. A single band of hybridization was observed at 46A on the right arm of chromosome II (Figure 7A). This is in contrast to the known position of trp, which has been located at 99C5-6 on chromosome III (Monte11 et al., 1985). The trp gene product has been localized by immunohistochemical methods to the retinula cell rhabdomeres of the fly (Monte11 and Rubin, 1989). As trp and trplare structurally related, we sought to compare their tissue-specific expression. We have observed that an antisense riboprobe, made using the AB 3.14/

We have identified a cDNA encoding a novel calmodulin-binding protein from Drosophilaand shown that this protein is structurally related and spatially expressed in a manner similar to the Drosophila trp gene product. It seems likely, therefore, that the trpl gene isalso involved in the phototransduction process.The signal transduction cascade in the fly’s photoreceptor cells has recently been reviewed (Ranganathan et al., 1991). Briefly, the pathway is initiated by light-activated rhodopsin, which in turn is believed to activate a phospholipase C via a G protein (Blumenfeld et al., 1985). The resulting production of inositol 1,4,5-trisphosphate (IPs) (Devary et al., 1987; lnoue et al., 1988; Bloomquist et al., 1988) increases intracellular Ca2+ levels by releasing Ca *+ from intracellular stores. By analogy with other invertebrates, it is this IP3-induced increase in intracellular free Caz+ that is believed responsible for both activation of the light-sensitive ion channel and light adaptation (Payne et al., 1986). However, in studying the effects of intracellular injection of Ca2+-chelating agents on the light-activated response of Limulus photoreceptors, Lisman and Brown (1975) concluded that Ca2+ could not be solely responsible for the light-activated response. Furthermore, Johnson et al. (1986) have implicated cGMP in activation of invertebrate photoreceptors. In the Drosophila trp mutant, the phenotype consists of an altered electroretinogram, in which the light-induced steady-state depolarization of the photoreceptor cells decays to baseline levels (Cosens and Manning, 1969; Pak, 1979; Minke, 1982). This inactivation of the receptor potential is observed at high light intensities, but not under dim light conditions. It seems likely, both from structural considerations and from the localization of the trp protein to the rhabdomeres, that the trp gene product is an integral membrane protein (Monte11 and Rubin, 1989). This, taken togetherwith theevidence indicatingthat trp mutants reduce intracellular Ca*+ (Kirschfeld and Vogt, 1980; Lo and Pak, 1981; Minke, 1982), suggests that the trp protein could be regulating the levels of free Cal+ in the photoreceptor cells, but at a point after the production of IP3 in the phototransduction cascade (Montell and Rubin, 1989).

The Structure That It Could

of the trpl Protein Suggests Be a Channel Protein

That the trpl gene product may be a membrane protein is suggested by its hydrophobicity profile as well as its size. The Western blot in Figure 1 shows that

trpl: A Novel 637

Figure cDNA

Drosophila

7. Chromosomal

Calmodulin-Binding

and Tissue

Protein

In Situ Hybridization

of trpl

(A) Hybridization in situ of the AB3.14 insert to Drosophila salivary gland polytene chromosomes. The main photomicrograph was taken using phase-contrast; the inset photograph shows the same region of thechromosome, using interference-contrast, to highlight the deposition of stain. A single band of hybridization (arrowhead) is observed at region 4bA on chromosome II. (B and B’) Spatial localization of the trpl gene transcript. Hybridization above background was seen only in those sections in which the antisense probe was used and only in head sections. fB) Interference-contrast photograph of a head section: R, retina (photoreceptor layer); L, lamina; M, medulla. (B’J Dark-field photograph of the same section showing the presence of silver grains only in the photoreceptor layer.

largecalmodulin-binding

proteins

(>I00

kd)arefound

almost exclusively in the membrane fraction. More importantly, both the trpland trp proteins are homologous in most of their putative transmembrane regions to the equivalent membrane-spanning regions of both Ca2+ and, to a lesser extent, Na+ channel proteins. It is therefore possible that both trp and trpl encode subunits of Ca2+ or nonspecific cation channels. The voltage-dependent Ca2+ and Na’ channel proteins consist of four homologous domains, each with six membrane-spanning regions (Noda et al., 1984,1986; Salkoff et al., 1987;Tanabeet al., 1987; Hui et al., 1991). K+ channel proteins, on the other hand, have a single domain, but form channels by the association of subunits into a tetrameric structure (Tempel et al., 1987; Butler et al., 1989). As the trpl and trp proteins apparentlyconsist of a single domain, it is assumed that they will also form tetramers. The absence of the series of basic amino acids in the S4 region of the trpl and trp proteins and the divergence in the SSI and SS2 regions between the trpl and Ca2+ channel proteins are suggestive of these channels being gated by some mechanism other than membrane potential. It seems likely, therefore, that both the trp and trpl proteins are subunits of non-voltage-gated plasma membrane channels. As the trp protein is believed to act after the production of IPx in the phototransduction cascade, comparison was made of the amino sequence of both the trpl and trp proteins with the IP3-sensitive Ca2+ channel (Furuichi et al., 1989). No homology was found, suggesting that these proteins are not directly activated by IP3. The Role of trp/ and frp in the Phototransduction Cascade In their study of the trpgene, Monte11 and Rubin (1989) mooted the possibility that trp encodes the lightsensitive ion channel. This possibility was dismissed by these authors, for it was argued that if trp were to encode the light-sensitive ion channel, then a null mutation at the trp locus should show no lightinduced photoreceptor response. However, mutants that produce no trp gene product continue to exhibit a transient, light-sensitive depolarization of the photoreceptor cells. The identification of the trpl gene allows for a reassessment of this interpretation. Given that the trpland trp genes are likely to encode plasma membrane channel proteins, these could represent different forms/subunits of light-sensitive ion channels. The phenotype of trp null mutants could then be explained in terms of the residual light-induced activation of the trpl channel. The presence of two light-activated channels is also consistent with the findings of Bacigalupo et al. (1986; Bacigalupo et al., 1987, Biophys. J., abstract) and Nagy (1991), who measured two light-activated conductances in Limulus photoreceptors. The Function of the Calmodulin-Binding in the trpl Protein Light activation of photoreceptor

Site(s) cells

can

be

mim-

icked by the injection of IP3, suggesting that release of intracellular Ca2+ is responsible for the initial activation of the light-sensitive ion channel (Payne et al., 1986). In recent whole-cell, voltage-clamp studies of Drosophila photoreceptors, it was shown that extracellular Ca2+ also enters the photoreceptors in response to light stimulus (Hardie, 1991). This influx of Ca2+ produces both positive and negative feedback on the photoreceptor currents, resulting in stimulusdependent activation, inactivation, and adaptation of phototransduction (Hardie, 1991; Ranganathan et al., 1991). That the trpl protein contains at least one, and possibly two, calmodulin-binding sites associated with the carboxy-terminal region of the protein makes it an ideal target for regulation by changes in intracellular free Ca2+ levels. Given that the trpl protein has six membrane-spanning regions, the calmodulin-binding site(s) should be on the internal face of the membrane. Hence the binding of calmodulin will be sensitive to any increase in intracellular Ca2+concentration. The interaction of calmodulin with proteins is usually associated with an allosteric change in the structure and hence regulation of the target protein. Similarly, the binding of calmodulin to one or both sites on the trpl protein might be expected to regulate its activity. It should be mentioned here that the possibility of a single polypeptide binding more than 1 calmodulin molecule is not without precedent. Both caldesmon (Wang et al., 1989) and phosphofructokinase (Buschmeier et al., 1987) have two distinct calmodulinbinding domains. If trpl encodes a cation-selective channel, then the binding of Ca*/calmodulin could act to gate thechannel. Ca2+/calmodulin might bind toand open thechannel in response to the IP3-generated increase in intracellular levels of Ca*+ (Payne et al., 1986). This would be reinforced by the subsequent influx of Ca2+ via the channel (Hardie, 1991). Another possibility is that Ca2+/calmodulin may bind to and inactivate the trpl channel. The presence of two putative calmodulin-binding sites in the trpl protein could mean that Ca2+/ calmodulin mediates both activation and inactivation of the channel, but at different intracellular free Ca2+ concentrations. As described above, the affinity of Ca2+/ calmodulin for different targets protein may be dependent upon the intracellular free Ca2+ concentration. If, in the trpl protein, the binding of Caz+/calmodulin to one site occurs at relatively low Cal+ concentrations, but at the other only at high Ca2+ levels, then Ca2+ could differentially activate and inactivate the trpl channel dependent on the free Cal+ concentration. If only trpl were inhibited by Ca2+/calmodulin, this would allow for both a steady-state (trp) and an adaptive (trpl) response to the activation of these channel proteins. Interestingly, this is the kind of light-induced response seen in invertebrate photoreceptors at high light intensities and in the trp mutants. Furthermore, in Limulus, the injection of EGTA into the photoreceptor cell enhances the lightinduced, nonadapting steady-statedepolarization, in-

trpl. A

Novel

Drosophila

Calmodulin-Binding

Protein

639

dicating that intracellular free Ca2+ is necessary adaptation (Lisman and Brown, 1975).

for

The Ankyrin-like Repeat Both the trpl and trp proteins contain an ankyrin-like repeat structure. It has been suggested (Lux et al., 1990) that the ankyrin-like repeats found in the Notch gene of Drosophila may function to link the Notch protein to the G protein p subunit encoded by the Enhancer of split gene (Hartley et al., 1988). It is possible that the ankyrin-like repeat motifs of the trp and trpl proteins likewise interact with another protein, perhaps to activate the channels. However, it is also possiblethat this motif acts likeankyrin itself, binding the trp and trpl gene products to the microtubular cytoskeleton and directing and anchoring the proteins to particular regions of the rhabdomere membranes. Although much remains to be learned about the role of the trp and trpl genes in phototransduction, the identification of trpl as another gene likely to be involved in photoreceptor physiology and one that is probably regulated by Ca*+/calmodulin allows for the reinterpretation of the phenotype of the trp mutants. The observed homology between the trpl, and hence trp, protein and the a subunit of the mammalian brain Ca2+ channel supports the idea that the trp protein is a subunit of a light-activated ion channel and that the trpl protein represents a subuhit of a second lightactivated channel. Unfortunately, no known mutations that affect the phototransduction cascade map near the chromosomal location of trpl, and so the various hypothesesoutlined hereare not immediately testable. However, studies are presently underway to isolate trpl mutants. The study of trpl mutants, alone and in combination with trp, should be of particular interest. Finally, we have shown that the use of calmodulin as a ligand probe to identify cDNAs that encode calmodulin-binding proteins is a useful technique for the identification of novel calmodulin-regulated proteins. Hopefully, the characterization of the other two transcripts identified in this study will serve to illuminate other Ca*+/calmodulin-regulated processes. Experimental

Procedures

Purification of Sheep Brain Calmodulin Sheep brain calmodulin was purified using a single step phenothiazine affinity chromatography column. Sheep brain homogenized in 20 mM Tris-HCI (pH 7.5) containing 1 mM CaC12 and 1 mM MgCI,, with a tissue-to-buffer ratio of 1:3 (w/v), was centrifuged at 20,008 x g for 30 min. The supernatant was clarified by pouring it through glass wool. Ammonium sulfate was added (0.66 g/ml), and the resulting suspension was left standing at 4OC for1 hr.Aftercentrifugationat20,OOO x gforU)min,theresulting pellet was resuspended in a small volume of homogenization buffer and exhaustively dialyzed against the homogenization buffer. The sample was then treated at 90°C for 5 min, and after chillingon ice, the resulting precipitatewas removed bycentrifugation. The clear supernatant was then applied to a 1.6 x 15 cm phenothiazine-Sepharosecolumn (Bio-Rad), and the column was washed with the same buffer. Elution of the calmodulin was achieved using the same buffer, but with 2 mM EGTA added

instead of the CaCI,. This pure by SDS-polyacrylamide

preparation was judged gel electrophoresis.

to be >95%

Radioiodination of Cahnodulin Radioiodination of calmodulin was achieved using the glucose oxidase-lactoperoxidase method (Marchalonis, 1969). One hundred micrograms of calmodulin in 50 pl of 0.2 M phosphate buffer (pH 7.2) mixed with 25 ~1 of 1 mM P-D-glucose was added to50 VI of rehydrated Enzymobeads reagent (Bio-Rad, Richmond, CA) followed by the addition of 20 pl (1 mCi) of Na ‘=I. After the reaction was complete (20 min at room temperature), the radioiodinated calmodulin was separated from unincorporated 125l by applying the reaction mixture to a column (1.5 x 45 cm) of Sephadex G-75 equilibrated in 40 mM Tris-HCI buffer (pH 7.5) containing 50 mM NaCI, 0.3 mM CaCl*, and 0.5 mM dithiothreitol. Approximately 45% of the radioiodine was covalently bound tothecalmodulin, resultinginaspecificactivityofapproximately 2 x 106 cpm per pg of protein. Calmodulin labeled in this way gave high levels of binding, with a sufficiently high signal-tonoise ratio toallow the identification of positive plaqueson filter lifts. Preparation of Fly Extracts and Western Blots Frozen Drosophila heads from the Oregon-R strain were used to produce either crude membrane fractions or cytosolic fractions, run on SDS-polyacrylamide gels, and Western blotted as described previously (Kelly, 1990). Probing of Western Blots and Filter lifts with Radioiodinated Calmodulin Filters were blocked for at least 30 min using TBS (50 mM TrisHCI [pH 7.51, 150 mM NaCI) containing 0.1 mM CaCI, and 5% (w/v) skim milk powder. Filterswere probed using a small volume of the same solution containing approximately 1 x 106 cpmlml of V-labeled calmodulin and incubated for4 hr at 4°C. This was followed by three 10 min washes in TBS, 0.1 mM CaCI,, after which the filters were dried and autoradiographed. Identification of Cahnodulin-Binding Proteins Clones in a Drosophila Head cDNA library The Drosophila head cDNA library in Lgtll (Itoh et al., 1985) was plated using the E. coli strain Y1090 to give approximately IO5 plaques per 150 mm plate. The plates were then incubated at 42’X for 4 hr, at which point nitrocellulose filters, which had been soaked in 10 mM isopropyl-p-u-thiogalactopyranoside and dried were placed on the surface of the agarose and incubated at 37OC overnight. The filters werethen blocked and probed with ‘*Uabeled calmodulin as described for the Western blots. Lysogens were prepared from these positive phage in the E. coli strain Y1089. The lysogens were grown at 42OC and induced with isopropyl-p-o-thiogalactopyranoside (Young and Davis, 1983). The lysates from these strains were then electrophoresed on SDS-polyacrylamide gels (8%), Western blotted, and probed with radioiodinated calmodulin. Molecular Analysis of the AB3.14, AB 3.9, and AB3.14/Z9 Clones All of the general molecular biology procedures, including Northern and Southern blotting, wereaccording to the methods described in Maniatis et al. (1982). The AB3.14/Z9 cDNA clone was sequenced, according to the method of Sanger et al. (1977), in both directions using a combination of restriction fragments subcloned into Ml3 and a number of synthetic oligonucleotides as primers for regions of the cDNA clones that were inaccessible using the standard Ml3 primers. The original 1.7 kb AB3.14 and 1.1 kb A63.9 clones were also completely sequenced, although certain sections where there was no ambiguity with the AB3.14/Z clone were not sequenced in both directions. Sequence Analysis The hydropathy profile cording to the method

of the trpl protein was determined acof Kyte and Doolittle (1982) using a win-

NWKMl 640

dow of 17 amino acids. transmembrane regions

The algorithm used to identify possible was that described by Klein et al. (1985).

Chromosomal In Situ Hybridization In situ hybridization of the cDNA clones to Drosophila polytene chromosomes was carried out according to the method of Engels et al. (1986). Biotinylated probes were prepared using a BRL nick translation kit according to the manufacturer’s directions. Tissue In Situ Hybridization Both sense and antisense “S-labeled riboprobes were synthe sized from the AB3.14/Z9 clone and used to hybridize in situ to 10 urn frozen sections of adult flies according to the method of lngham et al. (1985). Acknowledgments We would like to thank Ms. Rebecca Ramsbotham for technical assistance, Mr. M. Ladomery forassistancewith sequencing, and Dr. L. Salkoff for helpful comments. This work was supported in part by a grant from the ARC (to L. E. K.). A. B. was a recipient of an Australian Commonwealth Post Graduate Research Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

November

12, 1991; revised

January

13, 1992.

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The CenBank in this paper

Number

accession is M88185.

number

for the &p/sequences

reported

II