Correspondence to: Craig Montell, Department of Biological Chemistry 408WBSB, The Johns Hopkins University School of Medicine, 725 No. Wolfe St., Baltimore, MD 21205. Tel:(410) 955-1199 Fax:(410) 614-9573
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The light response in Drosophila photoreceptor cells is mediated by a series of proteins that assemble into a macromolecular complex referred to as the signalplex. The central player in the signalplex is inactivation no afterpotential D (INAD), a protein consisting of a tandem array of five PDZ domains. At least seven proteins bind INAD, including the transient receptor potential (TRP) channel, which depends on INAD for localization to the phototransducing organelle, the rhabdomere. However, the determinants required for localization of INAD are not known. In this work, we showed that INAD was required for retention rather than targeting of TRP to the rhabdomeres. In addition, we demonstrated that TRP bound to INAD through the COOH terminus, and this interaction was required for localization of INAD. Other proteins that depend on INAD for localization, phospholipase C and protein kinase C, also mislocalized. However, elimination of any other member of the signalplex had no impact on the spatial distribution of INAD. A direct interaction between TRP and INAD did not appear to have a role in the photoresponse independent of localization of multiple signaling components. Rather, the primary function of the TRP/ INAD complex is to form the core unit required for localization of the signalplex to the rhabdomeres.
Key Words: PDZ, Drosophila, TRP, INAD, phototransduction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A longstanding view of G proteincoupled signaling cascades is that they function through random collisions among receptors, G proteins, and downstream effectors. However, such a concept of highly mobile signaling molecules is inconsistent with the specificity inherent in G protein signaling. During the last decade, there has been a growing body of evidence to dispute a random collision model and to suggest that the components in G protein cascades might, in fact, be tightly coupled (for reviews see
A clear demonstration of a G protein signaling cascade that is organized into a supramolecular signaling complex, signalplex, has emerged from analyses of Drosophila phototransduction (for reviews see 90amino acid protein interaction motifs called PDZ domains (
PDZ motifs occur in a large variety of proteins and bind to a diversity of signaling, cell adhesion, and cytoskeletal proteins (for reviews see
A minimum of seven proteins binds directly to INAD. These include rhodopsin (
Despite extensive biochemical and genetic analyses of the signalplex, the determinants required in vivo for localization of INAD are not known. Candidate anchoring proteins include the light receptor (rhodopsin), ion channels (TRP and TRPL), and the cytoskeletal protein, NINAC. Here, we show that TRP and INAD share a reciprocal requirement for retention in the rhabdomeres. INAD was mislocalized in an age-dependent manner in strong trp alleles, or as a consequence of deleting the INAD binding site in TRP. In contrast to a previous report (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Generation of Wild-type and Mutant GST-TRP Fusion Proteins
Wild-type TRP consists of 1275 amino acids (1272 mutation is a deletion of the COOH-terminal four residues in TRP (12721275), and V1266D is a valine to aspartic acid substitution at residue 1266. To generate the
1272 and V1266D mutations in the glutathione S-transferase (GST)-TRP fusion proteins, we performed oligonucleotide-directed mutagenesis using a single-stranded TRP genomic DNA fragment (nucleotides 41726480) inserted between the EcoRV and EcoRI sites of pBluescript KS+ (pBSgTRPEK; Stratagene). The wild-type and mutated DNA fragments were amplified by PCR (nucleotides 60046108) and inserted between the EcoRI and XhoI sites of pGEX-5X-3 (Amersham Pharmacia Biotech) to create the short-tail fusions. The short wild-type (encoding TRP residues 12471275) and mutant constructs are referred to as pGEX-STRP, pGEX-STRP-
1272, and pGEX-STRP-V1266D. The constructs encoding the long-tail fusions were generated by transferring Smal/SalI fragments (trp nucleotides 53566480) from pBSgTRPEK into the pGEX-5X-2 vector (Amersham Pharmacia Biotech). The long wild-type (encoding TRP residues 10301275) and mutant constructs are referred to as pGEX-LTRP, pGEX-LTRP-
1272, and pGEX-LTRP-V1266D. The GST-TRP constructs, as well as GST-INAD (pGST-INAD vector;
cells and the proteins were expressed as described (Amersham Pharmacia Biotech).
Pull-down Assays
Fusion proteins were prepared from 20 ml E. coli cells transformed with pGST-INAD or the wild-type or mutant pGEX-STRP or pGEX-LTRP constructs described above. After inducing expression of the fusion proteins for 4 h with isopropyl-ß-D-thiogalactoside (IPTG), the cells were pelleted by centrifugation, resuspended in 2 ml PBSIT buffer (PBS containing CompleteTM enzyme inhibitor cocktail [Boehringer] and 1% Triton X-100), and lysed by sonication. The lysates were rotated at 4°C for 30 min to further solubilize the proteins, and cellular debris was removed by centrifugation at 14,000 rpm. Fusion proteins were purified from the supernatants with glutathione beads (Amersham Pharmacia Biotech). To perform the pull-down assays, 1 µg GST-TRP fusion proteins or 4 µg GST was coupled to 30 µl glutathione beads and incubated with an equal amount of 35S-INAD or extracts prepared from fly heads (10 fly head equivalents) in 0.5 ml PBSIT. [35S]methionine-labeled INAD was synthesized in vitro by coupled transcription/translation using the TNT kit (Promega) and pBS-INAD as the template. Fly head extracts were prepared by homogenizing 50 fly heads in 0.5 ml PBSIT buffer followed by centrifugation at 14,000 rpm for 30 min to remove debris. After incubation with the probes (35S-INAD or head extracts), the beads were pelleted by centrifugation and washed four times with PBSIT. The proteins were eluted from the beads with 20 mM glutathione, and the eluates were fractionated by SDS-PAGE. The gels containing 35S-INAD were dried and exposed to X-ray film. The proteins in the gels containing extracts from fly heads were subjected to Western blot transfer, the polyvinylidene fluoride (PVDF) membrane was stained with anti-INAD antibodies, and signals were detected using an enhanced chemiluminescence (ECL) kit (NEN Life Science Products).
Stability of TRP and INAD in Photoreceptor Cells
To determine the stability of TRP and INAD in vivo, we performed a pulse-chase experiment using 35S-methionine. 300 late second instar or early third instar w1118 larvae were washed with PBS and transferred to a bottle with a square Whatmann No. 3MM paper adhered to the bottom. After draining the residual PBS that was transferred with the larvae, 0.7 ml labeling mixture (0.1 mCi/ml 35S-methionine [> 1000 Ci/mmol; ICN Biomedicals], 0.5 M sucrose, and 2 mM of each of the following cold amino acids: Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Phe, Ser, Thr, Tyr, Val) was dispensed onto the paper. Adult flies <12 h after eclosion were transferred in groups of 50 flies to individual vials containing normal fly food media consisting of cornmeal, molasses, and brewer's yeast. Fly heads were harvested 1, 3, 5, and 9 d later, and were homogenized in PBSIT as described above.
TRP and INAD were immunoprecipitated using protein A beads coupled to 3 µl anti-TRP and anti-INAD antibodies. The beads were prepared by blocking 30 µl protein A-Sepharose beads (Sigma-Aldrich) with 3% BSA in PBSIT, incubating with the antibodies in 300 µl PBSIT for 1 h, and washing two times in PBSIT. The IgG-coupled beads were added to extracts prepared from 50 fly heads, and the total volume was increased to 1 ml with PBSIT. The beads were rotated for 2 h at 4°C, pelleted, and washed four times in PBSIT. Proteins were eluted from the pelleted beads by addition of 40 µl SDSsample buffer and fractionated by SDS-PAGE (17 fly head equivalents), and the 35S-labeled proteins were detected using Kodak BioMax MR film and a Kodak BioMax TransScreen-LE (low energy) intensifying screen. The exposed films were scanned into a computer, and the mean darkness values of the protein bands were determined using the histogram function available in the Adobe Photoshop 5.0 program. After the background darkness levels were subtracted, the value obtained with each time point was divided by that of the corresponding of 1.5-d group to obtain the relative radioactivities.
Generation of trp1272 Transgenic Flies
A 6480-nucleotide genomic fragment has been shown previously to fully rescue trp (1272 deletion created in pBSgTRPEK (see above). pBSgTRPEK includes an EcoRV/KpnI fragment spanning nucleotides 41726480. The EcoRV/KpnI fragment was excised from pBSgTRPEK-
1272 and introduced into pKgTRPEE. pKgTRPEE includes an EcoRI/EcoRV fragment (nucleotides 14171) of TRP genomic DNA inserted into the pHFK vector. The pHFK vector contains a gene for kanamycin selection and a polylinker flanked at both ends with NotI sites. The full-length trp
1272 genomic DNA was excised with NotI and introduced into NotI site of the ry+ P-element transformation vector pDM30 (
1272 DNA was injected into trpCM,ry embryos to generate P[trp
1272] transformant flies (referred to as trp
1272 in the text). To facilitate the immunostaining analyses, the w1118 mutation was introduced into the transgenic flies to eliminate the red screening pigment in the compound eye that causes autofluorescence.
Immunolocalizations
Hemisected fly heads were fixed with paraformaldehyde and embedded in LR White resin as described (
To perform the electron microscopy, fly heads were hemisected, fixed for 23 h in 2% paraformaldehyde, 2% glutaraldehyde, and 0.1 M sodium cacodylate (pH 7.4), washed three times in 0.1 M sodium cacodylate, postfixed for 2 h in 1% osmium tetroxide and 0.1 M sodium cacodylate, and washed two times for 5 min each in H2O. After dehydration in 50%, 70%, and 90% acetone, the heads were embedded in LR White resin. The tangential sections examined were cut at a depth of 35 µm from the surface of the eyes and viewed by transmission electron microscopy.
Electroretinogram Recordings
Electroretinogram (ERG) recordings were performed as described (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mislocalization of TRP in InaDP215 Is Age Dependent
The original allele of inaD, referred to as InaDP215 (
Drosophila compound eyes consist of 800 ommatidia, each of which contains eight photoreceptor cells (reviewed in
To address whether TRP is physically linked to INAD for targeting and/or retention, we examined the spatial distribution of TRP in photoreceptor cells from young and old flies. In wild-type flies, TRP was localized exclusively in the rhabdomeres regardless of their age (Fig 1 A). However, the spatial distribution of TRP was altered in InaDP215 in an age-dependent manner. During the late pupal period, TRP was restricted to the rhabdomeres in both wild-type and InaDP215 photoreceptor cells (Fig 1 A). In adult flies <1 d old, TRP was detected primarily in the rhabdomeres, whereas a lower but significant fraction was observed in the cell bodies (Fig 1 A). By 7 d after eclosion, the spatial distribution of TRP was severely disrupted in InaDP215. In these older flies, at least as much anti-TRP staining was found in the central matrix as was found in the rhabdomeres (Fig 1 A). The pronounced mislocalization of TRP in the older InaDP215 flies was specific, as other signaling proteins that function in phototransduction, such as the Rh1 rhodopsin (Fig 1 B;
|
The age-dependent mislocalization of TRP in InaDP215 suggested that interaction with INAD was required for retention of TRP in the rhabdomeres. Alternatively, if TRP had a short half-life and was continuously synthesized at a high rate in vivo, the mislocalization of TRP in older InaDP215 might reflect a requirement for INAD for targeting in older flies. To differentiate between these models, we considered whether TRP was long or short lived by performing pulse-chase experiments. TRP was labeled in vivo by feeding third instar larvae 35S-methionine. Immediately upon eclosion, the adult flies were fed fly food media, which was devoid of the radioactive amino acid, for 19 d. We found that the level of TRP decreased only 25% between days 1.5 and 9.5 (Fig 2A and Fig C). Moreover, the total level of TRP was relatively constant between days 1.5 and 9.5 (Fig 2 B). Thus, it appeared that TRP was long lived, and that a large proportion of the TRP protein in 59-d-old flies was synthesized in young flies. Consequently, the mislocalization of TRP in 7-d-old InaDP215 (Fig 1 A) appeared to be due to a defect in retention rather than in targeting to the rhabdomeres.
|
Spatial Distribution of INAD Is Dependent on TRP
INAD is required for proper localization of TRP, PLC, and PKC in the rhabdomeres (
|
PKC and PLC, but not Rh1 or NINAC, were also mislocalized in the cell bodies in young trp photoreceptor cells (Fig 3 B). In addition, PKC staining was detected in the central matrix. These results were consistent with former reports that PKC and PLC require INAD for normal localization (
TRP Binds Directly to INAD through the COOH Terminus
Most PDZ-containing proteins bind to their targets via a COOH-terminal sequence that conforms to an S/T-X-V/I motif or that consists of aromatic and/or hydrophobic residues (1272; Fig 4 A). To test the effects of these mutations, we performed pull-down assays using GST-fusion proteins and a 35S-labeled INAD probe. 35S-labeled INAD bound to the GST fusion proteins consisting of the COOH-terminal 246 or 29 residues from wild-type TRP (referred to as long and short tails, respectively; Fig 4A and Fig B). The short TRP fusion protein containing the V1266D mutation bound 35S-labeled INAD as well as wild-type, although the V1266D mutation did decrease the interaction between INAD and the longer TRP tail. However, the
1272 mutation completely disrupted the interaction with INAD in the context of either the long or short TRP tail (Fig 4 B). Thus, although there was some contribution of the internal S-X-V motif to the TRP/INAD interaction, the COOH-terminal four residues were essential for binding.
|
To exclude the possibility that posttranslational modifications of INAD might promote or interfere with binding to TRP, fly head extracts were also used in pull-down assays. INAD from wild-type fly head extracts bound to GST-TRP (Fig 4 C). In addition, INAD also interacted with TRPV1266D, although the amount of INAD that was pulled down was slightly reduced relative to that with wild-type GST-TRP (Fig 4 C). Deletion of the last four amino acids from TRP obliterated the interaction with INAD (Fig 4 C). Identical results were obtained with either the short (Fig 4 C) or long tails (data not shown). In parallel control experiments, all TRP tails failed to bind INAD from InaDP215 head extracts.
The missense mutation in PDZ3 of INADP215 could potentially disrupt interactions with one or more proteins in addition to TRP. To determine the specific role of the TRP/INAD interaction, we generated transgenic flies, P[trp1272], expressing TRP channels missing the last four amino acids, which are crucial for binding to INAD. It has been shown previously that the TRP protein declines in an age-dependent manner in InaDP215 (
1272. Based on Western blot analysis, the TRP protein level in <1-d-old trp
1272 was comparable to that in wild-type flies (Fig 5 A). In 7-d-old flies, the level of TRP decreased more than sixfold in trp
1272 compared with a less than threefold reduction in InaDP215 (Fig 5 A). The level of INAD protein did not decrease significantly in 7-d-old trp
1272 flies; however, there was a slight age-dependent reduction in the trp343 flies (Fig 5 B). The concentrations of other members of the signalplex, such as PLC and PKC, were also unchanged in 7-d-old trp
1272 (Fig 5 B). Thus, the mislocalization of INAD, PLC, and PKC in trp
1272 did not appear to be a secondary effect of protein degradation. Moreover, these results demonstrate that mislocalization of signaling components from the rhabdomeres does not obligatorily result in an increased turnover of the proteins. The concentrations of INAD, PLC, and PKC were decreased in trp343 (Fig 5 B). This effect appeared to be due to degeneration of the rhabdomeres, as the level of NINAC p174 was also reduced (Fig 5 B), even though it is not dependent on interaction with the signalplex for stability or localization (
1272 protein from transgenic flies could bind INAD, we performed pull-down assays using GST-INAD and extracts from wild-type and trp
1272 fly heads. As expected, wild-type TRP but not TRP
1272 from head extracts interacted directly with INAD (Fig 5 C).
|
Age-dependent Mislocalization of TRP and INAD in trp1272 Flies
To determine the requirement for the TRP/INAD interaction for localization of TRP and INAD, we stained sections of trp1272 compound eyes with antibodies to each of the two proteins. During the first 24 h after eclosion, a large proportion of the TRP
1272 staining was detected in the rhabdomeres (Fig 6 A). In addition, significant staining was detected in the cell bodies. By 7 d after eclosion, TRP
1272 was no longer concentrated in the rhabdomeres relative to the cell bodies (Fig 6 A). In parallel with the mislocalization and reduction of TRP
1272, INAD also displayed age-dependent alterations in spatial distribution in trp
1272 flies. The localization of INAD was mildly disrupted in young trp
1272 flies (Fig 6 C), but was normal in similarly aged norpA and inaC flies (Fig 3 A). This mild mislocalization did not appear to be due to degeneration, as the morphology of trp
1272 rhabdomeres was similar to that observed in wild-type, norpA, and inaC (Fig 6 E). By 7 d after eclosion, the concentration of INAD in the rhabdomeres of trp
1272 flies had declined dramatically (Fig 6 C). Given that the half-life of INAD was
5.5 d in vivo (Fig 2A and Fig C) and that the proportion of INAD remaining in the rhabdomeres of 7-d-old trp
1272 was barely detectable, it appeared that TRP was required for retention of INAD in the rhabdomeres.
|
The mislocalization of TRP and INAD in trp1272 flies was much less severe in flies maintained in the dark. In contrast to 7-d-old trp
1272 flies reared under a normal light/dark cycle, a high proportion of TRP and INAD localized to the rhabdomeres in 7-d-old dark-reared flies (Fig 6B and Fig D). The proportion of TRP and INAD in the rhabdomeres of these latter flies was similar to that observed in young (<1 d after eclosion) trp
1272 maintained under a light/dark cycle.
PLC and PKC were also mislocalized in old trp1272 flies; however, the time course and spatial distributions of PLC and PKC were distinct. In wild-type, PLC and PKC were localized specifically to the rhabdomeres (Fig 3;
1272 photoreceptor cells, a high proportion of the PKC was also detected in the rhabdomeres, although a significant concentration of PKC was observed in the cell bodies (Fig 7). By 7 d after eclosion, PKC was more uniformly distributed between the central matrix, the rhabdomeres, and cell bodies. The mislocalization of PLC in trp
1272 was more rapid than for PKC, and was already pronounced in young trp
1272. In older flies, the majority of the anti-PLC staining was found in the cell bodies. The mislocalization of TRP, INAD, PLC, and PKC in old trp
1272 flies was specific to those proteins that depend on the signalplex for localization, as there was no perturbation in the rhabdomere-specific distribution of Rh1 (Fig 7). Although INAD, PLC, and PKC were all mislocalized in trp
1272, the time course was slightly slower than was the case in trp343. In addition, INAD, PLC, and PKC were unstable in trp343 but not trp
1272. These differences are most likely due to rhabdomeral degeneration in trp343, as a rhabdomeral marker that does not depend on the signalplex for stability or localization, NINAC p174, decreases in an age-dependent manner in trp343 but not trp
1272 (Fig 5 B).
|
Normal Photoresponse in Young trp1272 Flies
The data presented above indicate that an important function of the TRP/INAD interaction is to retain both TRP and INAD in the rhabdomeres. If the primary function of the TRP/INAD association is to maintain these two proteins in the rhabdomeres, then young trp1272 flies would be expected to display a photoresponse similar to wild-type. According to this model, the photoresponse should be similar to wild-type, as the concentration of TRP and INAD in the rhabdomeres of young trp
1272 is nearly normal. Alternatively, if the primary role of linking TRP directly to INAD is to facilitate rapid signaling, then the photoresponse should be disrupted in young trp
1272 flies.
To address whether the INAD/TRP interaction has a primary role in the photoresponse, we performed ERG recordings. ERGs are extracellular recordings that measure the summed responses of all retinal cells to light. Exposure of wild-type flies to a bright orange-light stimulus results in a rapid corneal negative response because of influx of cations. Upon cessation of the stimulus, there is a rapid return of the maintained component to the baseline (Fig 8 A). We found that ERGs recorded from young trp1272 flies were similar to wild-type, although the amplitude of the maintained component was slightly reduced (Fig 8 A). In particular, and in contrast to young InaDP215, there was no delay in termination of the photoresponse (t1/3 = 89 ± 44 ms versus 1.55 ± 0.47 s; Fig 8 A). In InaDP215, exposure to a shorter light stimulus (0.7 versus 10 s) resulted in a less severe termination defect (t1/3 = 0.53 ± 0.13 s versus 1.55 ± 0.47 s) (Fig 8 B). Furthermore, in 7-d-old trp
1272, there was no apparent defect in termination (t1/3 = 138 ± 76 ms) of the photoresponse, although there was a pronounced decline in the amplitude of the maintained component (Fig 8, AC).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Requirement for INAD for Retention Rather Than Targeting of TRP
The INAD signalplex has emerged as a paradigm for understanding the composition and function of a G proteincoupled macromolecular assembly. We have shown previously that INAD is required for the normal localization of TRP (1272 flies, whereas in older flies, the spatial distribution of TRP was severely disrupted. These data suggested that INAD may be required for retention rather than targeting of TRP to the rhabdomeres. However, an alternative interpretation of these data is that those TRP molecules synthesized in young flies are targeted through an INAD-independent mechanism, whereas INAD is required for targeting of TRP synthesized in older flies. In support of the proposal that the INAD/TRP interaction was required for retention was the observation that TRP was long lived in vivo. TRP molecules synthesized before day 1.5 declined only
25% in concentration during the next 8 d. Thus, it appears that TRP is initially targeted to the rhabdomeres, and is subsequently mislocalized in the absence of a direct link to INAD.
TRP and INAD Form the Core Unit of the Signalplex
An intriguing question concerns the identification of proteins required for localization of INAD. The NINAC myosin III would appear to be an excellent candidate, as it binds INAD and is a putative molecular motor expressed in the photoreceptor cells. Nevertheless, NINAC is not required for localization of INAD (1272, in which the INAD binding site was deleted, the spatial distribution of INAD was disrupted in an age-dependent manner. These data, in combination with the findings that the half-life of INAD was
5.5 d, suggested that the mislocalization of INAD in trp mutant flies was due to a defect in retention.
In addition to a requirement for the TRP/INAD interaction for localization of TRP and INAD, elimination of trp or mutation of the INAD binding site in TRP led to an alteration in the spatial distribution of other proteins that require INAD for rhabdomere localization. These include PLC and PKC. Moreover, the mislocalization of PLC and PKC appeared to be more pronounced than that of INAD in young trp flies. One possibility is that these signaling proteins may also interact with TRP and depend on both TRP and INAD for localization. PKC appears to interact at least transiently with TRP, as TRP is a substrate for PKC. Thus, TRP and INAD appear to form the core unit that is required for localization of many of the signalplex components in the rhabdomeres.
The putative tetrameric structure of TRP may contribute to the stability of the TRP/INAD core unit, as each channel would have the potential to bind four INAD proteins. Although INAD was mislocalized in trp1272, there was no major impact on the localization of INAD in InaDP215 (
1272 than in InaDP215. The presumed tetrameric structure of TRP could enhance a weak interaction between INADP215 and TRP in vivo, which is not observed in pull-down assays using a monomeric TRP tail, as each channel would have the potential to bind four INAD proteins. The data presented here raise the possibility that other PDZ-containing scaffold proteins form similar core complexes in vivo with tetrameric ion channels. In support of this proposal are recent in vitro experiments indicating that there is a reciprocal requirement for localization of PSD-95 and the K+ channel, Kv1.4 (
A separate question that awaits further investigation concerns the mechanism underlying targeting of the signalplex to the rhabdomeres. Evidence has been presented that another PDZ-containing scaffold protein, PSD-95, is trafficked to the postsynaptic compartment after assembling on vesicles (
TRP/INAD Association May Facilitate Retention of Proteins during Membrane Turnover
The finding that TRP and INAD were long lived was surprising considering that there is very active turnover of the rhabdomeric membrane (1272 flies maintained in the dark. We suggest that a greater proportion of TRP and INAD was retained in the dark because of less turnover of the rhabdomeral membrane in the absence of light.
Direct TRP/INAD Interaction Is Not Required for the Photoresponse
In contrast to InaDP215, the ERG response in young trp1272 was similar to wild-type. The only significant ERG phenotype in trp
1272 flies was an age-dependent decrease in the amplitude of the maintained component. This defect was presumably due to mislocalization of TRP and INAD, as the amplitude of the maintained component gradually decreased in parallel with the mislocalization of TRP and INAD in older flies. Moreover, the termination of the photoresponse appeared normal even in old trp
1272 flies. This latter result was surprising, as PKC is mislocalized in old trp
1272 photoreceptor cells, and PKC is required for termination of the photoresponse (
1272. Given that PKC, TRP, and INAD have been reported to be present in about equimolar concentrations (
The defect in termination associated with InaDP215 (
To address the specific role of the TRP/INAD interaction, we mapped the INAD binding site and generated transgenic flies expressing a TRP derivative that did not associate with INAD. PDZ domains typically recognize COOH-terminal sequences consisting of an S/T-X-V/I motif or hydrophobic or aromatic residues (1272 obtained from transgenic fly head extracts failed to associate with INAD in pull-down assays. Mutation of an internal S-X-V motif (V1266D), which had previously been reported to abolish interaction between TRP and INAD in an overlay assay (
We conclude that the primary role of the direct interaction between TRP and INAD is not to facilitate rapid signaling. The apparently normal ERG in young trp1272 suggests that there is no defect in any aspect of the photoresponse. Rather, binding of TRP to INAD is critical for forming the core unit of the signalplex, which is necessary for retention of multiple signaling proteins in the rhabdomeres. This conclusion contrasts with the previous report, which concluded that INAD functions as a regulatory subunit of the TRP channel (
1272 indicate that the delayed termination associated with InaDP215 (
Multiple Functional Classes of INAD-binding Proteins
It appears that there are at least three classes of INAD binding proteins. The first class consists exclusively of TRP, as it is the only known INAD binding partner that is required for retention of INAD as well as of those INAD targets that depend on the signalplex for localization. However, there may be additional proteins that along with TRP and INAD comprise the core unit.
The second group includes two proteins, PLC and PKC, which rely on INAD for localization and stability. However, there is no reciprocal requirement for these proteins for retention of any other protein in the rhabdomeres. Mutation of the INAD binding sites in PLC (
The third class of INAD target proteins includes proteins such as rhodopsin, NINAC, and TRPL that are not dependent on INAD for localization in the rhabdomeres. We propose that the class I and II proteins, which depend on interaction with INAD for retention in the rhabdomeres, are constitutively bound to INAD, whereas the class III proteins may interact dynamically with INAD. As a consequence, only a subset of the class III proteins may bind to INAD at any given time. The observation that class III proteins do not depend on INAD for localization suggests that these INAD/target protein interactions have an alternative function, such as a direct role in the photoresponse. In support of this proposal, we have recently found that mutation of the INAD binding site in NINAC results in a pronounced delay in termination of the photoresponse (
![]() |
Footnotes |
---|
1 Abbreviations used in this paper: ERG, electroretinogram; GST, glutathione S-transterase; INAD, inactivation no afterpotential D; PKC, protein kinase C; PLC, phospholipase C; TRP, transient receptor potential.
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We thank Randall Shortridge for providing the anti-PLC antibodies, and Gulayse Ince for assistance with the Western blots.
This work was supported by grants EY08117 and EY10852 from the National Eye Institute (to C. Montell).
Submitted: 18 April 2000
Revised: 26 July 2000
Accepted: 7 August 2000
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|