©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The cDNA Sequence of Human Endothelial Cell Multimerin
A UNIQUE PROTEIN WITH RGDS, COILED-COIL, AND EPIDERMAL GROWTH FACTOR-LIKE DOMAINS AND A CARBOXYL TERMINUS SIMILAR TO THE GLOBULAR DOMAIN OF COMPLEMENT C1q AND COLLAGENS TYPE VIII AND X (*)

(Received for publication, May 17, 1995)

Catherine P. M. Hayward (§) John A. Hassell Gregory A. Denomme Richard A. Rachubinski Claudia Brown John G. Kelton

From the Departments of Pathology, Medicine, and Biochemistry and the Institute for Molecular Biology and Biotechnology, McMaster University, and the Canadian Red Cross Blood Transfusion Service, Hamilton Centre, Hamilton, Ontario, L8N 3Z5 Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Multimerin is a massive, soluble protein found in platelets and in the endothelium of blood vessels. Multimerin is composed of varying sized, disulfide-linked multimers, the smallest of which is a homotrimer. Multimerin is a factor V/Va-binding protein and may function as a carrier protein for platelet factor V. The cDNA for human multimerin was isolated from gt11 endothelial cell libraries using antibodies, and the isolated cDNA clones were used to obtain the full sequence. The full-length multimerin cDNA was 4.2 kilobase pairs. Northern analyses identified a 4.7-kilobase transcript in cultured endothelial cells, a megakaryocytic cell line, platelets, and highly vascular tissues. The multimerin cDNA can encode a protein of 1228 amino acids with the probable signal peptide cleavage site between amino acids 19 and 20. The protein is predicted to be hydrophilic and to contain 23 N-glycosylation sites. The adhesive motif RGDS (Arg-Gly-Asp-Ser) and an epidermal growth factor-like domain were identified. Sequence searches indicated that multimerin is a unique protein. Analyses identified probable coiled-coil structures in the central portion of the multimerin sequence. Additionally, the carboxyl-terminal region of multimerin resembles the globular, non-collagen-like, carboxylterminal domains of several other trimeric proteins, including complement C1q and collagens type VIII and X.


INTRODUCTION

Multimerin is a large, soluble protein (1, 2) stored within platelet alpha-granules (3) and endothelial cell Weibel-Palade bodies. (^1)Following activation of these cells, multimerin is released and binds to the cell surfaces of platelets(1, 2, 3, 4) , and endothelial cells,^1 and the extracellular matrix. In vivo, multimerin is restricted to megakaryocytes, platelets, and the endothelium and subendothelium of blood vessels, and it is not found in the plasma(3, 4) .^1 Recent studies have identified multimerin as a factor V/Va-binding protein. (^2)In resting platelet lysates, multimerin is complexed with factor V, and immunoelectron microscopy studies indicate that factor V and multimerin are stored together within platelet alpha-granules.^2 However, following platelet activation and the release of multimerin and factor V, these two proteins dissociate.^2 These findings suggest that multimerin may play a role in the storage and stabilization of platelet (but not plasma) factor V and also indicate separate functions for these proteins on activated platelets. The avid association of multimerin with activated platelets (1, 4) and endothelial cells^1 suggests that there may be other functions, possibly adhesive, for multimerin once it is released from intracellular stores.

Multimerin is one of the largest proteins found in platelets and endothelial cells, with most of its multimers exceeding a million daltons in size(1, 2, 3, 4) .^1 The variable molecular weight of multimerin is due to differences in the number of multimerin subunits comprising the protein(1, 2, 4) . Multimerin is highly glycosylated, with complex N-linked carbohydrate accounting for about one-third of its molecular mass(3) . It is synthesized as a p-170 protein (132-kDa polypeptide component) containing high mannose, N-linked carbohydrates, which are then converted to complex forms(3) . During biosynthesis, interchain disulfide bonds form to generate homotrimers and larger homomultimers(2, 3, 4) . Proteolysis of the subunits occurs (without disrupting the multimeric structure), leading to the stable p-155 subunit that is stored in platelets(1, 2, 3, 4) .

A number of parallels exist in the protein trafficking and storage of multimerin and von Willebrand factor. Multimerin resembles von Willebrand factor in its complex, disulfide-linked multimeric structure (2, 4) . However, unlike von Willebrand factor, which is assembled from dimers, the smallest multimer of multimerin is a 400-kDa homotrimer (2) . Both proteins are stored within the electron-lucent zone of platelet alpha-granules and within the Weibel-Palade bodies of endothelial cells(3) .^1 They are constitutively secreted as small multimers, and their intracellular stores are enriched in high molecular weight multimers(5) .^1 However, unlike von Willebrand factor, multimerin is not detectable in plasma and differs in its subunits size and glycosylation(1, 3, 4) .

In this report, we describe the isolation, sequencing, and deduced amino acid sequence of human endothelial cell multimerin cDNA. These studies identify multimerin as a unique protein, unrelated to von Willebrand factor, with RGDS, EGF-like, and coiled-coil domains, and a carboxyl-terminal region that resembles the trimeric, carboxyl-terminal globular domains of complement C1q and collagens type VIII and X.


MATERIALS AND METHODS

cDNA Libraries

Human endothelial cell cDNA libraries in gt11 were obtained from Clontech (5` stretch human endothelial cDNA library; Palo Alto, CA) and additional human umbilical vein endothelial cell libraries (VII-91-4 and VII-91-5) were a generous gift from J. Evan Sadler (6) (St. Louis, MO).

Screening of gt11 cDNA Libraries

Libraries were screened for clones (7) expressing the multimerin protein using both monoclonal and polyclonal anti-multimerin antibodies (1) (1:1000 dilution), alkaline phosphatase-conjugated secondary antibodies (1:10,000 dilution, Promega, Madison, WI), and nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (Sigma) for detection. All of the clones identified by antibody screening were immunoreactive with both monoclonal and polyclonal multimerin antisera. Following cloning and sequencing of the initial isolates, the most 5` clone was labeled and used to rescreen the gt11 library for full-length cDNA clones(8, 9) .

Lambda DNA was purified from plate lysates(10) , digested with EcoRI, and the multimerin cDNA clones were subcloned into the EcoRI site of pGEM 7Zf+ (Promega).

PCR^3 Amplification of Inserts

The multimerin cDNA inserts ingt11 were amplified using PCR and primers specific for gt11, as described previously (11) . The inserts were subcloned and their flanking gt11 sequences were used to determine the orientation of the multimerin cDNAs(12) . These clones were also used for sequencing across an internal EcoRI site, using multimerin-specific sequencing primers.

DNA Labeling

Multimerin cDNA inserts were labeled with [alpha-P]ATP, using a random primer labeling kit (U. S. Biochemical Corp.), for use in library screening and Northern blotting. The 5` EcoRI fragments of the full-length multimerin cDNA (mm17) and of mm4 were used for Northern analyses.

DNA Sequencing

Double-stranded sequencing of overlapping clones was performed using manual (dideoxy sequencing with Sequenase, U. S. Biochemical Corp.) and automated sequencing of Qiagen miniprep DNA (Qiagen, Chatsworth, CA). Automated DNA sequencing was performed by the Central Facility of the Institute for Molecular Biology and Biotechnology, McMaster University, using an Applied Biosystems (model 373A) automatic DNA sequencer. Sequencing was done using dyedeoxy terminator technology with cycle sequence Taq according to the manufacturer's instructions. Primers used for sequencing included M13 universal forward and reverse primers. Multimerin-specific primers were used to fill in gaps in the aligned sequence of exonuclease III-deleted clones. PCR-derived clones were used only for studies of orientation and for sequencing across the internal EcoRI site.

Data Analyses

Sequence analyses and alignments were performed using the MacVector and Assemblylign programs (Eastman Kodak Co.) and PC Gene (IntelliGenetics, Inc., Mountain View, CA). Data bank searches (NCBI at National Library of Medicine, Bethesda, MD: nonredundant PDB+SwissProt+SPupdate+PIR+GenPept+GPupdate)were performed using the BLASTP algorithm. Further alignments of homologous sequences were performed using the Clustal program (DNA Star Ltd., London) and a PAM 250 matrix. Analyses for coiled-coil structures were performed by Dr. Seth Darst, Rockefeller Institute, using the program PEPCOIL (Genetics Computer Group, Madison, WI).

Protein Purification and Sequencing

Multimerin was purified from outdated platelet concentrates by affinity chromatography, as described(1) . 15 µg of the purified protein was used for preparative SDS-polyacrylamide gel electrophoresis (reduced) and transblotted onto a polyvinylidene difluoride membrane (Bio-Rad), following the manufacturer's instructions. The 155-kDa multimerin subunit was localized using Ponceau Red, excised from the membrane, and used to obtain internal amino acid sequence data. Protein digestion (lysylendopeptidase C), peptide separation, and amino acid sequencing were performed by the Harvard Microchemistry Facility (Cambridge, MA) using an ABI 477A protein sequencer with a 120A PTH-AA analyzer.

Northern Analyses

Northern analysis was performed as described(9, 13) . RNA was isolated from first passage endothelial cells,^1 platelets (washed platelet pellet (1) from 30 ml of whole blood), and from resting and PMA-treated Dami cells (3) using TRIzol (Life Technologies, Inc.). 20 µg of total RNA was loaded per lane (1% agarose gels), and RNA markers (Promega) were used to determine the transcript sizes. Hybridization was performed using P-labeled multimerin cDNA (mm4 and mm17) to analyze the expression of multimerin in cultured cells and in multiple tissues (multiple tissue Northern; Clontech), using high stringency washes, as recommended by the manufacturer. Multimerin expression was compared with actin (cDNA probe supplied by Promega) as the control.


RESULTS AND DISCUSSION

Molecular Cloning and Sequencing of Full-length Multimerin cDNA

The deduced protein sequence of multimerin was investigated by cloning and sequencing of the multimerin cDNA. As previous studies indicated synthesis of multimerin by endothelial cells,^1 human endothelial cell cDNA libraries in gt11 were chosen for these studies. Because the NH(2) terminus sequence of multimerin was found to be blocked, antibodies were used for screening. The full-length multimerin cDNA was predicted to contain a 3.6-kbp open reading frame, based upon the 132-kDa polypeptide component of the multimerin precursor(3) .^1

Screening of the 300,000 plaques-forming units from the Clontech endothelial cell cDNA expression library yielded seven multimerin immunoreactive clones (mm1-7). Sequencing of the 5` and 3` ends of mm4, mm5, and mm7 identified overlap in their sequences, and all three isolates were recognized by both monoclonal (JS-1) and polyclonal multimerin antisera. All of the gt11 libraries screened were constructed using EcoRI adapters. Digestion of mm2 and mm5 liberated two EcoRI fragments, indicating the presence of at least one internal EcoRI site in the multimerin cDNA. To identify the orientation of the expressed clones in gt11 and the number of internal EcoRI sites, the complete mm5 and mm7 inserts were amplified using primer sites in the arms (11, 12) and subcloned into pGEM. Sequencing of the PCR-amplified inserts identified the orientation of the fragments and indicated that only one internal EcoRI site was present in mm5. A 3` 729-bp fragment containing a polyadenylation signal site and terminating in a poly(A) tail (Fig. 2) was identified. The most 5` clone identified by antibody screening (mm4, 1.4 kbp) terminated at the internal EcoRI site. Exonuclease III deletions of the overlapping clones (mm4 and mm7) were created to fully sequence the cDNA fragments in both directions (Fig. 1).



Figure 2: Nucleotide sequence and deduced amino acid sequence of human endothelial cell multimerin. An in-frame stop codon in the 5`-untranslated region is underlined. An internal EcoRI site is indicated. The 3`-untranslated region contains a polyadenylation signal and terminates in a poly(A) tail. Amino acids 368-376 were confirmed by sequencing an internal peptide fragment of multimerin. The location of the most probable signal peptide is indicated. Potential N-linked glycosylation sites (dot) are noted, and cysteine residues are underlined. RGDS and EGF-like domains are indicated. A partial EGF-like domain, lacking the first cysteine of the EGF concensus sequence CXCXXXXXGXXC, is also indicated.




Figure 1: Multimerin sequencing strategy. Arrows indicate the direction and distance of sequencing. Clones subcloned as PCR fragments are indicated with ``pcr'' in their name. The consensus clone length was 4212 bp.



The radiolabeled mm4 cDNA was used to screen the libraries VII-91-4 and VII-91-5 for a full-length cDNA clone. Two clones were isolated that contained additional 5` sequence: mm11 (isolated from VII-91-4; 2.1- and 0.75-kbp EcoRI fragments) and mm17 (isolated from VII-91-5; 3.7-kbp EcoRI fragment). The first isolate, mm11, was used to obtain additional sequence 5` of mm4, using multimerin-specific primers. Subsequently, exonuclease III deletions were created from mm17, and the region 5` (and overlapping) of mm4 and mm11 was fully sequenced in both directions. Sequencing of the 5` end of mm17 identified an in-frame stop codon prior to an initiation codon. This reading frame was consistent with the multimerin fusion proteins that had been identified by antibody screening of the gt11 library(12) . These findings indicated that a complete cDNA sequence had been obtained. The sequence derived from the overlapping clones was 4212 bp in length.

The sequence of the human endothelial cell multimerin cDNA is shown in Fig. 2. The open reading frame is preceded by a 71-bp noncoding region containing an in-frame stop codon. The initiation ATG conforms to Kozak's consensus sequence for initiation (14) and is followed by an open reading frame of 1228 codons and a 3`-noncoding region of 454 bp containing a polyadenylation signal and a poly(A) tail.

Deduced Protein Sequence of Multimerin and Homology with Other Proteins

The putative protein encoded by the multimerin cDNA was compared with amino acid sequence data obtained from purified platelet multimerin (Fig. 2). High confidence sequence data were obtained from an internal peptide fragment of platelet multimerin. These sequence data were in complete agreement with the predicted protein sequence (amino acids 368-376), indicating that the cDNA sequenced encodes multimerin.

The multimerin cDNA encodes a protein of 1228 amino acids with a calculated molecular mass of 138 kDa (Fig. 2). Analysis for the signal peptide cleavage site, using Prosite (PC Gene), indicated that the most probable cleavage site was between amino acids 19 and 20. The protein, minus the signal peptide, has a predicted molecular mass of 136 kDa, which is in close agreement with the 132-kDa nonglycosylated precursor identified by metabolic protein labeling studies of Dami cell (3) and endothelial cell multimerin.^1 Kyte-Doolittle plots indicated that the protein was hydrophilic, consistent with the partitioning of multimerin into the aqueous phase of Triton X-114 platelet extracts(1) . Alignment of the multimerin polypeptide sequence to itself using MacVector and a PAM250 matrix failed to identify significant internal repeats within the multimerin protein sequence.

Analysis of the multimerin protein for functional domains using MacVector and PC Gene identified the adhesive motif RGDS (amino acids 186-189) (15, 16) and an EGF-like (17, 18) domain (amino acids 1065-1076). Consensus sequences for a tyrosine sulfation site (amino acid 1038) (19, 20) and an asparagine hydroxylation site (21) (amino acid 1058) were identified adjacent to the EGF-like domain. The protein contained 23 potential N-glycosylation sites(22, 23, 24, 25) , which is in close agreement with the 17-21 sites predicted by N-deglycosylation of endothelial cell and Dami cell multimerin(3) .^1 The RGDS site was located in an unglycosylated region of the molecule with a high local flexibility score (MacVector, Karpus-Schulz analysis).

Search of the NCBI data banks using the BLASTP algorithm and BLOSUM 62 matrix indicated that multimerin was a novel protein. Assessment of the high scoring homologous sequences identified similarities between multimerin and a large number of proteins that contain EGF-like domains. The highest scoring elements were Xotch (26) and its homologues in other species. These homologues are transmembrane proteins, containing multiple EGF-like domains, and are important for neurogenic development. The highest scoring human proteins with homology to the EGF-like domain of multimerin included TAN-1(27) , a homologue of Xotch and Notch(28) , fibroblast proteoglycan core protein (29) , and coagulation factors IX (30, 31, 32) and X(33, 34, 35) . Fig. 3(upper panel) shows the comparison alignments for the EGF-like domain of multimerin. An additional region of homology between multimerin and Xotch, Notch, and TAN-1 proteins was identified spanning amino acids 265 and 303 of multimerin (Fig. 3, lower panel). This region of multimerin contains three cysteine residues but lacks the first cysteine in the EGF-like consensus sequence CXCXXXXXGXXC. This domain is homologous with EGF-like domains in Xotch, Notch, and TAN-1 and in proteoglygcan core proteins.


Figure 3: Comparison alignments of multimerin with proteins containing EGF-like domains. The upper panel shows the comparison of the EGF-like domain in multimerin with the EGF-like domains of the highest scoring, homologous protein sequences. The lower panel is a comparison of a domain of multimerin with similarity to EGF-like domains in Xotch, Notch, and the human homologue TAN-1. This region of multimerin lacks the first cysteine residue of an EGF-like consensus sequence. Boxed residues indicate consensus residues, and an asterisk indicates the conserved residues in an EGF-like motif.



A number of proteins, including the rod-like tail of many myosin heavy chains (from a variety of species and tissue types)(36, 37, 38) , macrogolgin(39) , and the tpr oncogene(40) , showed homology with regions in the central portion of multimerin between amino acids 317-1024. These homologous proteins are known to contain coiled-coil structures. Multiple sequence comparisons using the Clustal program (DNA Star) and a PAM 250 matrix revealed one region where residues were conserved between multimerin (amino acids 476-498) and many myosin heavy chain sequences (Fig. 4). The similarities between multimerin and a variety of coiled-coil proteins suggested that there could be coiled-coil structures within multimerin. This possibility was investigated using sequence analysis the program PEPCOIL. The multimerin polypeptide sequence contained regions of high probability for coiled-coil structures in the region of the protein that was similar to other coiled-coil proteins. The probable coiled-coil structures were located between amino acids: 317-375, 400-445, 668-738, and 818-873 (Fig. 5).


Figure 4: Comparison alignments of multimerin with the rod-like tail of myosin heavy chains. Boxed residues indicate consensus residues.




Figure 5: The probability of coiled-coil structures in multimerin. A number of highly probable coiled-coil regions are identified between amino acids 317-873.



An additional region of significant homology was observed in the carboxyl terminus of the multimerin sequence. This region of multimerin was found to resemble the trimeric, carboxyl-terminal, non-collagen-like, globular domain of several other proteins. These included the A, B, and C chains of human and mouse complement C1q protein(41, 42, 43, 44, 45, 46) and human alpha-1 collagens type VIII (47) and X (47, 48, 49, 50, 51) . The protein sequence comparisons for this domain indicate conservation of hydrophobic and uncharged residues (Fig. 6). Electron microscopy studies of these homologous proteins have shown that their carboxyl-terminal domains form a globular-shaped head(53, 54, 55) . In C1q, the globular domain is assembled from the carboxyl-terminal regions of an A, a B, and a C chain(56) . The globular domains of C1q and collagens type VIII and X are implicated in protein interactions. This domain in the complement C1q protein is known to interact with the Fc portion of IgG and other complement activator molecules, leading to complement activation(56) . In collagen type VIII, which is a heterotrimer of alpha-1 and alpha-2 chains, the COOH-terminal globular domain is implicated in the assembly of a mesh-like structure of collagen type VIII molecules(54) . Collagen type X, a homotrimeric protein, contains a similar COOH-terminal globular domain(47, 48, 49, 50, 51) . In collagen type X, the carboxyl-terminal globular domains associate to form a hexagonal mesh of collagen molecules(55) .


Figure 6: Comparison alignments of multimerin with the COOH-terminal globular domains of complement C1q A, B, and C chains and human alpha-1 collagens type VIII and X. Boxed residues indicate consensus residues.



A schematic summary of the various multimerin domains is shown in Fig. 7. Based upon the multimerin constructs that contained the epitope recognized by the monoclonal antibody JS-1, the JS-1 epitope was localized to the region containing amino acids 961-1139, which includes the EGF-like domain and a portion of the COOH-terminal globular domain.


Figure 7: Multimerin protein domains. Domains identified by protein analysis and the location of the monoclonal antibody JS-1 epitope are shown.



Because of the many similarities observed in the multimeric structures and protein trafficking of multimerin and von Willebrand factor(2, 4) ,^1 we had anticipated that there could be similarities in their amino acid sequences. However, von Willebrand factor was not identified in the data bank searches for homologous proteins, and direct comparison of the von Willebrand factor (57) and multimerin protein sequences failed to identify significant homologies. While the propolypeptide region has been postulated to be involved in the targeting of von Willebrand factor to Weibel Palade bodies(52, 58) , the lack of similarity in the sequences of multimerin and von Willebrand factor suggest that other factors must account for their similar trafficking in platelets and endothelial cells.

Our earlier studies of multimerin biosynthesis identified a p-170 precursor protein, containing high mannose-linked carbohydrate(3) . The 132-kDa polypeptide component of this p-170 multimerin precursor is in close agreement with the size of the deduced amino acid sequence minus its signal peptide (136 kDa). Based on these data, we have designated the multimerin precursor protein (minus the signal sequence) as promultimerin. During biosynthesis, the N-linked carbohydrates on promultimerin are converted to complex forms to produce a p-186 protein in endothelial cells and a p-196 protein in Dami cells(3) .^1 The site(s) of proteolytic cleavage that produce the p-155 multimerin subunit (105-kDa polypeptide component) (3) that is stored within platelets (1) and endothelial cells^1 await identification. Because the EGF-like domain, globular C1q-like domain, and JS-1 epitope are located at the carboxyl end of the protein sequence, we postulate that the mature protein is produced by cleavage in the NH(2)-terminal region.

Analyses of Multimerin RNA

Northern analysis of cultured cells and human tissues identified a 4.7-kb transcript using the multimerin cDNA probe (Fig. 8). Previous metabolic protein labeling studies indicated that multimerin is synthesized by endothelial cells^1 and by Dami cells (3) (a megakaryocytic cell line) after stimulation with PMA. The multimerin transcript was identified in cultured endothelial cells, Dami cells, and platelets. Comparison of resting and PMA-stimulated Dami cells indicated increased expression of the multimerin mRNA following stimulation of these cells with PMA. The identification of the multimerin transcript in platelets indicates that endogenous biosynthesis by megakaryocytes is the source of platelet multimerin. Comparison of multiple tissues identified the highest expression of multimerin in placenta, lung, and liver, three highly vascular tissues (Fig. 8). The detection of multimerin mRNA in vascular tissues is in agreement with the localization of multimerin in vascular endothelium in situ using immunohistochemistry.^1


Figure 8: Northern analysis of multimerin expression in cultured cells and in human tissues. The expression of multimerin (upper panels) and actin (lower panels) mRNA in isolated human cells and in human tissues. The left panels show cultured cells and platelets (20 µg of total RNA/lane), and the right panels are a multiple tissue Northern (Clonetech; 2 µg of poly(A) RNA/lane). Resting (-PMA) and PMA-treated (+PMA) Dami cells are shown for comparison. A 4.7-kb transcript is identified in endothelial cells, PMA-treated Dami cells, platelets, and in lung, placenta, and liver (highly vascular tissues).



The data presented in this report confirm that multimerin is a unique protein. Further studies are required to define the precise roles of multimerin's RDGS, coiled-coil, EGF-like, partial EGF-like, and C1q-like domains and to determine how the multimerin subunits are assembled into the large, disulfide-linked multimers. The coiled-coil and globular domains are likely sites for interchain associations. We postulate that the RGDS, EGF-like, and C1q-like domains will prove to be important sites for the interaction of multimerin with other proteins. Knowledge of the functions of these domains and of multimerin's tertiary structure may provide insights into the molecular mechanisms that control hemostasis. Additional clues may be provided by investigations of individuals who are deficient in multimerin.


FOOTNOTES

*
These studies were funded by a grant from the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U27109[GenBank].

§
Centennial Fellow of the Canadian Medical Research Council. To whom correspondence should be addressed: Dept. of Pathology, McMaster University, HSC 2N32, 1200 Main St. W, Hamilton, Ontario, L8N 3Z5 Canada. Tel.: 905-521-2100 (ext. 3373); Fax: 905-577-0198.

^1
C. P. M. Hayward, J. G. Kelton, R. H. Stead, T. E. Warkentin, and T. J. Podor, submitted for publication.

^2
Hayward, C. P. M., Furmaniak-Kazmierczak, E., Cieutat, A.-M., Moore, J. C., Bainton, D. F., Nesheim, M. E., Kelton, J. G., and Ct, G. (1995) J. Biol. Chem.270, in press.

^3
The abbreviations used are: PCR, polymerase chain reaction; PMA, phorbol 12-myristate 13-acetate; kbp, kilobase pair(s); bp, base pair(s); EGF, epidermal growth factor.


ACKNOWLEDGEMENTS

The technical assistance of Jane C. Moore and Zhili Song is gratefully acknowledged. The authors thank Drs. William Sheffield and Aled Edwards (McMaster University) for their helpful discussions and Dr. Seth Darst (Rockefeller Institute) for performing the analysis for coiled-coil structures.


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