©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Interaction of Calmodulin with Clathrin-coated Vesicles, Triskelions, and Light Chains
LOCALIZATION OF A BINDING SITE (*)

(Received for publication, May 9, 1994; and in revised form, November 7, 1994)

Ursula M. Pley (1) Beth L. Hill (2) Christine Alibert (2) Frances M. Brodsky (3) Peter Parham (1) (2)(§)

From the  (1)Departments of Microbiology and Immunology and (2)Cell Biology, Stanford University School of Medicine, Stanford, California 94305 and the (3)Department of Pharmacy and Pharmaceutical Chemistry, School of Pharmacy, and the Department of Microbiology and Immunology, School of Medicine, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The binding of clathrin-coated vesicles, clathrin triskelions, and free clathrin light chains to calmodulin-Sepharose was compared. When isolated from bovine brain, all three components bound to calmodulin-Sepharose in the presence of calcium and could be eluted by its removal. In contrast, coated vesicles and triskelions isolated from bovine adrenal gland did not bind to calmodulin-Sepharose, although the free light chains from adrenal gland bound as effectively as those from brain. As distinct isoforms of the clathrin light chains are expressed by brain and adrenal gland, these results implicate the clathrin light chains as the calmodulin-binding component of coated vesicles and triskelions. Furthermore, the insertion sequences found in the neuron-specific isoforms, although not necessary for the binding of free clathrin light chains to calmodulin, must facilitate the interaction of heavy chain-associated light chains with calmodulin. Recombinant mutants of LCa, with deletions spanning the entire sequence, were tested for binding to calmodulin-Sepharose. Those mutants retaining structural integrity, as assessed by the binding of a panel of monoclonal antibodies, exhibited varying amounts of calmodulin binding activity. However, deletion of the carboxyl-terminal 20 residues abolished calmodulin interaction. Thus, the carboxyl terminus of LCa appears to constitute a calmodulin-binding site. Peptides corresponding to the carboxyl terminus of LCa or LCb inhibited the interaction of the light chains with calmodulin, suggesting that this region forms the calmodulin-binding site of both LCa and LCb. The carboxyl-terminal peptides of LCa and LCb inhibited the interaction of light chains with calmodulin 10-fold less effectively than a calmodulin-binding peptide derived from smooth muscle myosin light chain kinase, but much more effectively than a calmodulin-binding peptide derived from adenylate cyclase. This comparison places the clathrin light chain-calmodulin interaction within the physiological range seen for other calmodulin-binding proteins.


INTRODUCTION

In eucaryotic cells, clathrin-coated vesicles are the agents for receptor-mediated transport of macromolecules between membrane-bound compartments. Formation of a coated vesicle is initiated by the binding of adaptors to receptors, thereby forming the inner layer of the protein coat. The outer layer is formed by the polymerization of clathrin upon the clustered adaptors, giving the coat its characteristic appearance of a polygonal lattice. Vesiculation, driven by the polymerization of clathrin, is followed by disassembly of the clathrin coat, allowing fusion of the vesicle with its target membrane (reviewed in (1) and (2) ).

The monomeric form of clathrin is the triskelion, a three-legged structure that can participate in many cycles of assembly and disassembly. Each leg of the triskelion consists of a heavy chain bound to a light chain(3, 4) . Mammalian cells express two homologous light chains, LCa and LCb, in contrast to the single light chain of yeast (5, 6, 7, 8, 9) . Additional heterogeneity in mammalian light chains arises from alternate mRNA splicing, which in neurons produces isoforms of LCa and LCb that have additional insertion sequences of 30 and 18 amino acids, respectively(5, 6) .

Central to coated vesicle function is the regulation of clathrin disassembly. Studies in vitro show that disassembly is effected by the cytosolic heat shock protein Hsc70 and is enhanced by the presence of calcium ions(10, 11, 12, 13) . Hsc70 appears to act through interaction with a binding site on the clathrin light chains that lies adjacent to a site for binding calcium(11, 14, 15) . Further evidence for the involvement of calcium in clathrin function is the observation that calmodulin, a cytoplasmic mediator of calcium-regulated processes, binds to bovine brain clathrin-coated vesicles and to clathrin light chains in a calcium-dependent manner(9, 16, 17, 18, 19) . The binding of calmodulin to clathrin triskelions has been visualized by electron microscopy, but not by affinity chromatography, and when clathrin heavy chains are immobilized on nitrocellulose, they, too, bind calmodulin(17, 20, 21) .

The experiments reported here compare the binding of calmodulin to clathrin-coated vesicles, triskelions, and light chains from tissues expressing the different light chain isoforms and define a binding site for calmodulin in the carboxyl-terminal region of the clathrin light chains.


MATERIALS AND METHODS

Coated Vesicle Preparation and Clathrin Purification

Clathrin-coated vesicles were prepared from six bovine brains or 2 pounds of adrenal glands as described previously (22) using a modified isolation buffer in which magnesium chloride and dithiothreitol were omitted. For the preparation of adrenal gland clathrin-coated vesicles, the protease inhibitors aprotinin (5 µg/ml), leupeptin (5 µg/ml), and pepstatin (7 µg/ml) were included, in addition to phenylmethylsulfonyl fluoride, in the isolation buffer. Triskelions were extracted from clathrin-coated vesicles and purified according to Näthke et al.(15) . Triskelions were boiled for 10 min to release the clathrin light chains (23, 24) . LCa and LCb were separated on a DEAE-cellulose column as described by Parham et al.(25) .

Light Chain Mutagenesis

Construction of the LCa mutants Delta26-56, Delta22-96, and Delta162-243 was reported previously(15) . LCa mutant Delta98-161 was produced by removing the ClaI-HindIII fragment from a full-length cDNA clone of bovine brain LCa(6) . Filling in of the restriction site overhangs and blunt-ended ligation then eliminated the codons for Leu-Glu (numbers refer to those of the original bovine LCa sequence(6) ). LCa mutant Delta2-21 was generated by deleting the NcoI-NaeI fragment from a full-length cDNA clone of bovine brain LCa(6) , followed by filling in and blunt-ended ligation. This resulted in a mutant LCa lacking residues Ala^2-Ala. LCa mutant Delta2-21,26-56 is a combination of LCa mutant Delta2-21 and Delta26-56. LCa mutants Delta188-243, Delta196-243, Delta204-243, and Delta224-243 were constructed from a bovine LCa cDNA clone in pUC18 containing the entire coding sequence of the major neuron-specific isoform. Stop codons were introduced at the desired locations through site-directed mutagenesis by overlap extension using the polymerase chain reaction(26) . Mutations were verified by DNA sequencing.

Preparation of Recombinant Mutant Light Chain

Mutant and wild-type LCa were cloned into either the pET11d or pET21d vector (Novagen) and expressed in Escherichia coli strain BL21(DE3) by isopropyl-beta-D-thiogalactopyranoside induction. Cell extracts containing recombinant LCa proteins were prepared by lysing cells in 50 mM Tris, pH 8.0, 70 mM EDTA, 9.5% sucrose, 0.06% Triton X-100, and 0.16 mg/ml lysozyme in the presence of the protease inhibitors aprotinin, leupeptin, pepstatin, and phenylmethylsulfonyl fluoride as described under ``Coated Vesicle Preparation and Clathrin Purification.'' Debris was removed from the cell lysates by a 10-min centrifugation at 50,000 times g. The supernatants were boiled and centrifuged to give a light chain-containing supernatant.

Radiolabeling of Rabbit Anti-mouse IgG F(ab`)(2) Fragment

50 µg of rabbit anti-mouse IgG F(ab`)(2) fragment was iodinated using 1 mCi of NaI (100 mCi/ml; Amersham Corp.) and one bead of IODO-BEADS (Pierce) to a specific activity of 14 µCi/µg.

Calmodulin-Sepharose Affinity Chromatography

To assay the interaction of light chains and mutant LCa proteins with calmodulin, 0.2 ml of settled calmodulin-Sepharose 4B resin (400 µg of calmodulin) was pre-eluted with 20 mM Tris, pH 7.8, 100 mM NaCl, 20 mM 2-mercaptoethanol, and 2 mM EGTA (buffer A) and re-equilibrated in 20 mM Tris, pH 7.8, 100 mM NaCl, 20 mM 2-mercaptoethanol, and 1 mM CaCl(2) (buffer B). Light chains (10-20 µg) in 0.5 ml of buffer B were incubated with the calmodulin-Sepharose resin for 1 h at 4 °C under moderate agitation on a rotating platform. The resin was then poured into a 1-ml tuberculin syringe to make a column from which the following fractions were collected: 0.5 ml of supernatant from the incubation mixture, 10 washes of 1 ml each of buffer B, and four elutions of 0.5 ml each of buffer A. The protein in each fraction was precipitated by 5% trichloroacetic acid and then analyzed by 12.5% SDS-PAGE (^1)(27) and Western blotting(28) . LCa was detected using monoclonal antibody CVC.7 (29) or X16(30) , and LCb was detected using monoclonal antibody LCB.1 or LCB.2(31) . The secondary antibodies were anti-mouse IgG-alkaline phosphatase for LCa and LCb proteins (Boehringer Mannheim) and radiolabeled rabbit anti-mouse IgG F(ab`)(2) fragments for recombinant LCa and LCa mutant proteins. As the negative control, a resin of Sepharose CL-4B (Pharmacia Biotech Inc.) with no calmodulin conjugated was used. For quantitation of recombinant LCa and LCa mutant proteins binding to calmodulin-Sepharose, Western blots were exposed to Phosphor Imager screens (Molecular Dynamics, Inc.) and read on a PhosphorImager SF (Molecular Dynamics, Inc.), and protein band intensities were determined using the ImageQuant software package (Molecular Dynamics, Inc.). The supernatant from the incubation mixture and the first and the tenth wash fractions were counted as material not bound to calmodulin-Sepharose. The eluted fractions were counted as material bound to calmodulin-Sepharose. The percentage of mutant light chain bound to calmodulin was calculated by dividing the radioactivity of the material eluted from the calmodulin columns by the total radioactivity of the bound and the unbound material from the calmodulin columns. Assays of clathrin-coated vesicle binding to calmodulin-Sepharose were done in 10 mM MES, pH 6.5, 100 mM KCl, 0.1% bovine serum albumin, 0.02% NaN(3), and either 2 mM EGTA or 1 mM CaCl(2). The buffer originally used for the binding of triskelions to calmodulin-Sepharose was 0.5 M Tris, 50 mM MES, pH 6.2, 0.25 mM MgCl(2), 0.5 mM EGTA, 1.5 mM CaCl(2), and 0.02% NaN(3). Triskelions were eluted from calmodulin-Sepharose with 0.5 M Tris, 50 mM MES, pH 6.2, 0.25 mM MgCl(2), 2 mM EGTA, and 0.02% NaN(3). Upon review of this report, the binding of brain triskelions to calmodulin-Sepharose was also performed in buffers containing 100 mM NaCl in addition to all other components listed above, and the results were identical.

Radiolabeling of LCa and LCb

200 µg of purified bovine brain LCa or LCb was radioiodinated using 1 mCi of NaI (100 mCi/ml) and one bead of IODO-BEADS per reaction for 15 min in buffer containing 25 mM Tris, 0.1 M NaCl, pH 7.0. I-Labeled light chain and unincorporated NaI were separated by gel filtration on a 0.8 times 16-cm Sephadex G-25 column equilibrated in 25 mM Tris, 0.1 M NaCl, and 1 mg/ml bovine serum albumin, pH 7.0. LCa and LCb were radiolabeled to specific activities of 12.9 and 2.89 µCi/µg, respectively.

Light Chain-Calmodulin Interaction in Presence of Peptides

Peptides (see Table 1) were synthesized by Research Genetics (Huntsville, AL) and desalted using Sep-Pak C(18) cartridges (Waters Associates) using published procedures(32) . After purification, the concentrations of the peptide preparations were determined using the Pierce micro-bicinchoninic acid assay according to the manufacturer's instructions. Assays were performed using 1.5 µg of I-labeled LCb or 0.5 µg of I-labeled LCa and 100 µl of settled resin of a mixture of calmodulin-Sepharose (Pharmacia Biotech Inc.) and Sepharose CL-4B. Assays contained 15 µg of calmodulin. Peptides at 1-, 10-, 100-, and 1000-fold molar excesses over light chain were coincubated with the radiolabeled light chains and the calmodulin-Sepharose/Sepharose CL-4B mixture for 1 h. Fractions were obtained as described above and analyzed by counting aliquots in triplicate on a Pharmacia Biotech 1260 Multigamma II counter.




RESULTS

Brain, but Not Adrenal Gland, Clathrin-coated Vesicles and Triskelions Bind to Calmodulin-Sepharose

The interaction of calmodulin with clathrin-coated vesicles (assembled clathrin) and purified triskelions (unassembled clathrin) was assessed in separate experiments. Clathrin-coated vesicles were isolated from bovine brains and tested for binding to calmodulin conjugated to Sepharose 4B. In the experiments using clathrin-coated vesicles, the presence of disassembled clathrin in the vesicle preparations was reduced by centrifugation at 100,000 times g. After discarding the supernatant that contained unassembled clathrin (triskelions), the pelleted clathrin-coated vesicles were resuspended just prior to use. The resin was incubated with clathrin-coated vesicles in the presence of Ca, extensively washed in the presence of Ca, and then eluted with buffer containing EGTA and no Ca. Fractions obtained from the chromatography were first centrifuged at 100,000 times g to separate assembled (clathrin-coated vesicles) from disassembled (triskelion) clathrin and were then analyzed separately for the presence of clathrin polypeptides using Western blots and a mixture of monoclonal antibodies specific for LCa, LCb, and the clathrin heavy chain.

This experiment shows that bovine brain clathrin-coated vesicles bind to calmodulin-Sepharose and can be eluted in a form that still sediments at 100,000 times g (Fig. 1A, leftpanel). 50% of the input material bound to the calmodulin column, and 10% of that was sedimentable following elution. The binding was dependent upon calmodulin as shown by the control experiment using unconjugated Sepharose (Fig. 1A, rightpanel).


Figure 1: Binding of brain clathrin-coated vesicles (A) and triskelions (B) to calmodulin. A, the leftpanel shows binding to calmodulin-Sepharose, whereas the rightpanel shows the negative control binding to Sepharose alone. Lanes1 and 2 show 124 µg of pelleted and nonpelleted brain clathrin-coated vesicles, respectively. An equivalent amount of pelleted vesicles was incubated with each resin. The unbound material was centrifuged, and the pellet is shown in lane3 and the supernatant in lane4. The resin was washed 10 times in the presence of Ca. In lanes5 and 6 are the pellet and the supernatant, respectively, obtained by centrifugation of the combined first and second washes. Similarly, lanes7 and 8 are the pellets and supernatants, respectively, obtained from the combined ninth and tenth washes. After washing, the resins were eluted with EGTA. The first two fractions eluted were combined and centrifuged; lanes9 and 10 show the pellet and the supernatant, respectively. Samples were analyzed by SDS-PAGE and Western blotting using a mixture of monoclonal antibodies against the clathrin heavy chain (TD.1), LCa (CVC.7), and LCb (LCB.2). The ratio of clathrin heavy chain to clathrin light chains may not seem constant in all lanes for several reasons. First, the Western blotting method employed was not intended to be quantitative. Second, the experiment analyzed intact clathrin-coated vesicles (in the lanes showing pelleted material) as well as clathrin-coated vesicles that disassembled and perhaps partially degraded during the duration of the experiment (in the lanes showing material of the supernatants). The positions of prestained molecular mass markers are indicated by the horizontallines to the left of the panels (from top to bottom): phosphorylase b (106 kDa), bovine serum albumin (80 kDa), ovalbumin (49.5 kDa), carbonic anhydrase (32.5 kDa), soybean trypsin inhibitor (27.5 kDa), and lysozyme (18.5 kDa). B, equivalent amounts of clathrin triskelions (lane6) were used to assess binding to calmodulin-Sepharose (lanes 1-5) and to Sepharose (lanes 7-11). After removal of unbound triskelions (lanes1 and 7), the resin was washed 10 times in the presence of Ca. Lanes2 and 8 show the first wash fraction, and lanes3 and 9 show the last wash fraction. Then, the resin was eluted with EGTA. Lanes4 and 10 show the combined first and second eluted fractions, and lanes5 and 11 show the combined third and fourth eluted fractions. The clathrin subunits were detected by silver staining of the SDS-polyacrylamide gel. Molecular mass markers were as described for A.



A similar analysis of clathrin triskelions derived from bovine brain clathrin-coated vesicles was performed. Buffer conditions were those that stabilize triskelions, and the samples for chromatography were centrifuged at 100,000 times g to remove aggregates prior to the incubation with the calmodulin-Sepharose. As observed for clathrin-coated vesicles, triskelions bound to calmodulin-Sepharose, but not Sepharose, in a Ca-dependent fashion (Fig. 1B).

When similar experiments were performed using clathrin-coated vesicles and triskelions isolated from bovine adrenal glands, no binding to calmodulin-Sepharose was observed (Fig. 2). For triskelions, the known differences between the preparations isolated from brain and adrenal gland are the presence of insertion sequences in the neuronal isoforms of LCa and LCb. These results suggested that the light chains could be involved in the interaction of calmodulin with brain clathrin-coated vesicles and triskelions and that the neuronal insertion sequences may play a role in these interactions.


Figure 2: Binding of adrenal gland clathrin-coated vesicles (A) and triskelions (B) to calmodulin. A, the left panel shows binding to calmodulin-Sepharose, whereas the rightpanel shows the negative control binding to Sepharose alone. Lanes1 and 2 show 100 µg of pelleted and nonpelleted adrenal clathrin-coated vesicles, respectively. An equivalent amount of pelleted vesicles was incubated with each resin. The unbound material was centrifuged, and the pellet is shown in lane3 and the supernatant in lane4. The resin was washed 10 times in the presence of Ca. In lanes 5 and 6 are the pellet and the supernatant, respectively, obtained by centrifugation of the first wash. Similarly, lanes7 and 8 are the pellets and supernatants, respectively, obtained from the tenth wash. After washing, the resins were eluted with EGTA. The first two fractions eluted were combined and centrifuged; lanes9 and 10 show the pellet and the supernatant, respectively. The observation of a small amount of heavy chain in lane9 was not reproducible. Samples were analyzed by SDS-PAGE and Western blotting using a mixture of monoclonal antibodies against the clathrin heavy chain (TD.1), LCa (CVC.7), and LCb (LCB.1). Molecular mass markers were as described for Fig. 1A. B, equivalent amounts of clathrin triskelions (lane6) were used to assess binding to calmodulin-Sepharose (lanes 1-5) and to Sepharose (lanes 7-11). After removal of unbound triskelions (lanes1 and 7), the resin was washed 10 times in the presence of Ca. Lanes 2 and 8 show the first wash fraction, and lanes3 and 9 show the last wash fraction. Then, the resin was eluted with EGTA. Lanes4 and 10 show the combined first and second eluted fraction, and lanes5 and 11 show the combined third and fourth eluted fractions. Samples were analyzed as described for A. Molecular mass markers were as described for Fig. 1A.



Isolated Brain and Adrenal Gland Forms of LCa and LCb Bind Calmodulin

Free light chains obtained from brain and adrenal gland triskelions were assessed for their binding to calmodulin-Sepharose. As previously shown by Lisanti et al.(18) , brain LCa and LCb bind well to calmodulin-Sepharose, and in our experiments, the binding was nearly complete (Fig. 3). Adrenal gland LCa and LCb also bound to calmodulin-Sepharose (Fig. 3), a result contrasting with that obtained for adrenal gland clathrin-coated vesicles and triskelions. As adrenal gland light chains lack neuronal insertion sequences, their binding cannot involve these sequences. The results from the binding of clathrin-coated vesicles, triskelions, and free light chain suggest the hypothesis that the calmodulin-binding site is formed by another part of the light chain molecule, but is modified by the presence of a neuronal insertion sequence.


Figure 3: Interaction of brain (A) and adrenal gland (B) clathrin light chains with calmodulin. In both A and B, the upperpanel shows the binding of LCa, and the lowerpanel shows the binding of LCb. A, 20-µg aliquots of the brain LCa and LCb preparations (lane 1) were used as input. Light chains were incubated with calmodulin-Sepharose, and the unbound material (lane 2) was removed. The calmodulin-Sepharose was then washed 10 times in the presence of Ca. Lanes 3-7 show successive pairs of the wash fractions. The resin was eluted four times with buffer containing EGTA (lanes 8-11). Samples were analyzed by SDS-PAGE and Western blotting with monoclonal antibodies against LCa (CVC.7) and LCb (LCB.2). B, input aliquots of adrenal gland LCa and LCb (lane1) were used for binding to Sepharose (lanes 2-6) and calmodulin-Sepharose (lanes 7-11). After coincubation of light chain and resin, material that was not bound to the resins was removed (lanes2 and 7), and the resins were then washed 10 times with Ca-containing buffer. Lanes 3 and 8 show the first wash fractions, and lanes4 and 9 show the tenth wash fractions. Then, the resins were eluted with buffer containing EGTA. Lanes5 and 10 show the combined first and second eluted fractions, and lanes6 and 11 show the combined third and fourth eluted fractions. Samples were analyzed by SDS-PAGE and Western blotting with monoclonal antibodies against LCa (CVC.7) and LCb (LCB.1).



COOH Terminus of LCa (Amino Acid Residues 224-243) Is Important for Interaction with Calmodulin

Ten deletion mutants of the major neuronal form of LCa were constructed. These deletions cover the entire LCa sequence (Fig. 4). To assess the structures of the LCa mutants, they were first compared with wild-type LCa for their recognition by eight LCa-specific monoclonal antibodies and polyclonal antisera on Western blots and by radioimmunoassay. Previous analysis mapped the epitopes recognized by the antibodies within the LCa sequence, and they are distributed throughout the length of LCa(11, 31, 33) . Wild-type LCa and all LCa mutants except mutant Delta162-243 reacted as predicted from the presence of antibody epitopes in the amino acid sequences. In contrast, LCa mutant Delta162-243 reacted weakly with five of the six antibodies tested recognizing epitopes in the NH(2) terminus and the heavy chain-binding domain, regions that are not deleted in the mutant. Thus, the deletion in mutant Delta162-243 grossly affects the conformation of LCa, whereas the other deletion mutants appear to fold sufficiently to retain antibody epitopes.


Figure 4: Cartoon showing linear organization of neuronal form of clathrin light chain LCa into structural and functional domains. Beneath the cartoon are line diagrams of the two LCa isoforms and the deletion mutants (mt) used in this study. Light gray box, conserved region; white wide striped box, Hcs70-binding site; dark gray box, calcium-binding site; white narrow striped box, heavy chain-binding site; black striped box, neuronal insert; black box, calmodulin-binding site.



To identify a region of LCa important for calmodulin binding, the deletion mutants were analyzed for interaction with calmodulin-Sepharose (Fig. 5). The calmodulin binding activity was calculated by dividing the radioactivity of the material eluted from the calmodulin column by the radioactivity of the total material (bound and unbound) recovered from the calmodulin column. The COOH-terminal truncation mutants Delta162-243, Delta188-243, Delta196-243, Delta204-243, and Delta224-243 did not bind calmodulin. In contrast, the LCa mutants carrying deletions in the NH(2) terminus or the heavy chain-binding domain exhibited calmodulin binding activity comparable to or exceeding that of wild-type recombinant LCa. These findings suggest that residues 224-243 at the carboxyl terminus of LCa are responsible for the calmodulin binding activity. The smallest COOH-terminal deletion that abolished calmodulin binding was that of mutant Delta224-243. As the serological analysis showed that the overall conformation of LCa mutant Delta224-243 was not disrupted, we hypothesize that residues 224-243 constitute a calmodulin-binding site.


Figure 5: Interaction of recombinant LCa and mutant LCa proteins with calmodulin-Sepharose (triplicate determinations are shown by filled bars) and Sepharose (clear bars). After incubation of mutant LCa proteins with resin, unbound material was removed, and the resin was washed 10 times with buffer containing Ca. LCa protein in these fractions was counted as not bound to the resin. Then, the resin was eluted with EGTA, and the ``bound'' protein was recovered. The yaxis shows the percentage of light chain bound as calculated from the ratio of bound protein and total protein (bound plus unbound). LCa and LCa mutant (mt) proteins were detected by Western blotting using monoclonal antibodies against LCa (CVC.7 and X16). The amount of protein in each band was quantitated as described under ``Materials and Methods.'' Black bar, calmodulin 1; light gray bar, calmodulin 2; dark gray bar, calmodulin 3; white bar, Sepharose.



Peptides Spanning Residues 224-243 of LCa and LCb Inhibit Light Chain-Calmodulin Interaction

The properties of the LCa deletion mutants identify residues 224-243 in the carboxyl-terminal region as a site of interaction with calmodulin. We therefore examined whether synthetic peptides derived from this LCa sequence and the homologous region of LCb (Table 1) inhibited the binding of I-labeled light chain to calmodulin-Sepharose. To assess the relative affinities of the clathrin light chain peptides for calmodulin, two peptides with known binding affinities for calmodulin, peptide SMMLCK derived from smooth muscle myosin light chain kinase (K(D) = 1 nM) (34, 35) and peptide AC derived from adenylate cyclase (K(D) = 580 nM)(36, 37) , were tested for their capacity to inhibit the binding of clathrin light chains to calmodulin in this assay (Table 1).

Four peptides spanning residues 224-243 of either LCa (LCa20 and LCa23) or LCb (LCb20 or LCb23) inhibit the calcium-dependent binding of I-LCb to calmodulin-Sepharose, whereas a peptide corresponding to residues 158-175 of the neuronal insertion sequence of LCa (nLCa) had no effect (Fig. 6B, upperpanel). Peptide SMMLCK was the most potent inhibitor of I-LCb binding to calmodulin, whereas peptide AC was ineffective. The light chain-derived peptides LCa20, LCa23, LCb20, and LCb23 are within an order of magnitude as effective inhibitors as peptide SMMLCK of the binding of I-LCb to calmodulin-Sepharose; we estimate their affinity constants for calmodulin to be 10 nM. To assess the importance of the sequence of the inhibitory peptides, as opposed to their amino acid composition, a ``scrambled'' peptide (LCa23sc) having the same length and composition as LCa23 but a different sequence was synthesized (Table 1). The LCa23sc peptide inhibited I-LCb binding to calmodulin-Sepharose, but with a potency 10-fold less than that of the LCa23 peptide (Fig. 6B, upperpanel). Thus, there appears to be contributions to calmodulin binding from both the sequence and the composition of the peptide inhibitors.



Figure 6: Competition for binding of I-labeled LCa (A) and I-labeled LCb (B) to calmodulin-Sepharose by light chain peptides. The upper and lowerpanels show the results for different peptides. I-Labeled LCa (0.5 µg) or I-labeled LCb (1.5 µg) was incubated with peptides at different molar ratios ranging between 1:1 and 1000:1 and calmodulin-Sepharose. After incubation, material that did not bind to the resin was removed, and the resin was washed six times with Ca-containing buffer. Radioactivity obtained in these fractions was counted as protein ``not bound'' to calmodulin-Sepharose. Then, the resin was eluted with EGTA, and the radioactivity recovered in these fractions was counted as protein bound to the calmodulin-Sepharose. The results are plotted as the percentage of light chain bound in the presence of peptide relative to that bound in the absence of peptide. Upper panels: black circle, LCa20; white circle, LCa23; multiplication sign, LCb20; black square, LCb23; black triangle, LCa23sc; white square, nLCa; white triangle, peptide SMMLCK; small dotted black square, peptide AC. Lower panels: white circle, LCa23; black triangle,LCa23sc; dotted black square, LCa12; dotted black diamond, LCa11; dotted white square, LCa12+11.



As a further test of the specificity of the inhibitory sequence, peptides corresponding to the amino-terminal (LCa12) and carboxyl-terminal (LCa11) halves of peptide LCa23 were synthesized (Table 1). The LCa11 peptide had no effect, whereas the LCa12 peptide gave a slight inhibition of I-LCb binding to calmodulin-Sepharose (Fig. 6B, lowerpanel). An analogous set of experiments assessed the capacity of the synthetic peptides to inhibit the binding of I-LCa to calmodulin-Sepharose, and the results are comparable to those obtained for the binding of I-LCb to calmodulin-Sepharose (Fig. 6A).

These studies with synthetic peptides support the assignment of residues 224-243 of LCa and LCb as a site of direct interaction with calmodulin. This site has a similar sequence in the two light chains: 15 of the residues are identical, and conservative substitutions are found at the remaining 5 (Table 1). Comparison of light chain peptide inhibition with well characterized calmodulin-binding peptides suggests that the affinity for the clathrin light chain-calmodulin interaction is within the range of other calmodulin-binding proteins and thus of potential physiological importance(37) .


DISCUSSION

Previous investigations have created a picture in which clathrin heavy and light chains are seen to play highly complementary roles. The characteristic structures of the triskelion and the clathrin coat are a function of the heavy chain. In contrast, the light chains contribute little to the appearance of clathrin structures, but provide an array of regulatory motifs implicated in the control of clathrin function (38) . Light chains are the target for phosphorylation, Hsc70 binding, and calcium binding.

Further evidence supporting this image of the light chains is contributed by the study described here; the carboxyl-terminal region of the light chains is demonstrated to be critical for interaction with calmodulin and in all likelihood forms the site of calmodulin binding. Calmodulin binding is the first activity mapped to the carboxyl-terminal region of the light chains that has been noted for its conservation between LCa and LCb(5, 6, 7) . Consistent with the conservation in sequence, we find that all four forms of free light chain, as represented by the neuronal and adrenal gland forms of LCa and LCb, bind to calmodulin affinity columns in a calcium-dependent fashion, confirming and extending the previous findings of Linden et al.(16) , Lisanti et al.(18) , Merisko et al.(21) , and Moskowitz et al.(17) .

Although assignment of the calmodulin-binding site to residues 224-243 is primarily based upon the analysis of deletion mutants of LCa, the circumstantial evidence derived from binding studies with intact light chains, inhibition studies with synthetic peptides, and the conservation of sequence indicate that the homologous region of LCb is also the calmodulin-binding site of that light chain.

Many cytoplasmic proteins bind calmodulin. A common motif in their calmodulin-binding sites is a basic amphiphilic alpha-helix(37) . The amino acid sequences of LCa and LCb are not predicted, however, to form such structures; instead, the light chains are, like calmodulin, characteristically acidic polypeptides. Within the calmodulin-binding region, aspartate 225 and proline 240 are predicted to perturb the basic nature of the site and alpha-helix formation, respectively. Although 4 basic and 7 hydrophobic residues are found within the region, they are spaced differently than in sequences known to bind calmodulin with high affinity(39) . Thus, it is possible that LCa and LCb bind calmodulin with structures distinct from a basic amphiphilic alpha-helix, precedent for which has been found in another calmodulin-binding protein, the -subunit of skeletal muscle phosphorylase kinase(40) . Moreover, amphiphilic peptides that are composed entirely of D-amino acids are able to bind calmodulin, demonstrating another permissible deviation from naturally occurring amphiphilic alpha-helices as a calmodulin-binding motif(41) . Furthermore, the light chain conformation is influenced by interaction with the heavy chain, which may affect the accuracy of predictions based solely upon light chain primary structure.

Free LCa and LCb are not the physiologically active forms of clathrin light chains, and assessment of the functional significance of their binding to calmodulin therefore requires an understanding of calmodulin interaction with triskelions and clathrin-coated vesicles. We find that brain triskelions and coated vesicles bind to calmodulin, whereas those isolated from adrenal gland do not. The clathrin heavy chain isolated from brain can bind calmodulin, whereas the heavy chain isolated from adrenal glands has not been analyzed(20, 21) . Therefore, it is unknown whether tissue-specific differences between the heavy chains exist. However, the known structural difference between the triskelions from these two tissues is the presence of neuron-specific insertion sequences in the light chains, suggesting that these sequences, which do not affect calmodulin binding of the free light chain, affect the conformation or accessibility of the calmodulin-binding sites on the light chains and possibly the heavy chain in triskelions and clathrin-coated vesicles. The insertion sequences in the light chains are close to the proposed calmodulin-binding site and are suitably situated to perform such modification. Alternatively, if differences between the heavy chains from the two tissues exist, unique features of the adrenal gland heavy chain might prevent adrenal gland triskelions from binding to calmodulin.

Differential interaction with calmodulin is the first assay of potential functional importance to distinguish the tissue-specific isoforms of the clathrin light chain. That calmodulin binds to neuronal clathrin but not to the clathrin of other cell types points to the calmodulin interaction serving a neuron-specific function. Potential candidates are the recycling of synaptic vesicle membrane proteins and the transport of triskelions along axons to the nerve terminals as both triskelions and calmodulin are transported to the synapse in the slow component SCb(42, 43) .


FOOTNOTES

*
This work was supported by grants from the American Cancer Society and the National Institutes of Health (to P. P.), by National Institutes of Health Grant GM38093 (to F. M. B.), by National Science Foundation Grant MCB-9118638, and by a grant from the Pew Charitable Trusts (to F. M. B.). 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.

§
To whom correspondence should be addressed: Dept. of Cell Biology, Fairchild Bldg., Stanford University, Stanford, CA 94305-5400. Tel.: 415-723-6224; Fax: 415-723-8464.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid.


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