CSMD1 is a novel multiple domain complement-regulatory protein highly expressed in the central nervous system and epithelial tissues

In this study, we describe the identification and in vitro functional activity of a novel multiple domain complement regulatory protein discovered based on its homology to short consensus repeat (SCR)-containing proteins of the regulators of complement activation (RCA) gene family. The rat cDNA encodes a predicted 388-kDa protein consisting of 14 N-terminal CUB domains that are separated from each other by a SCR followed by 15 tandem SCR domains, a transmembrane domain, and a short cytoplasmic tail. This protein is the homolog of the human protein of unknown function called the CUB and sushi multiple domains 1 (CSMD1) protein. A cloning strategy that incorporates the two C-terminal CUB-SCR domains and 12 of the tandem SCR repeats was used to produce a soluble rat CSMD1 protein. This protein blocked classical complement pathway activation in a comparable fashion with rat Crry but did not block alternative pathway activation. Analysis of CSMD1 mRNA expression by in situ hybridization and immunolabeling of neurons indicates that the primary sites of synthesis are the developing CNS and epithelial tissues. Of particular significance is the enrichment of CSMD1 in the nerve growth cone, the amoeboid-leading edge of the growing neuron. These results suggest that CSMD1 may be an important regulator of complement activation and inflammation in the developing CNS, and that it may also play a role in the context of growth cone function.     

Transcript map of the 8p23 putative tumor suppressor region

Cancers of the head and neck, prostate, liver, and bladder exhibit minimal regions of deletion within chromosomal band 8p23 that either overlap or map very close to one another. We previously refined a minimal region of deletion in squamous cell carcinomas to a 112-kb interval within 8p23. There seems to be only a single gene within this region that is expressed in normal upper aerodigestive tract epithelium. This candidate for the squamous cancer suppressor, CUB and sushi multiple domains-1 (CSMD1), extends into the minimal regions of deletions defined for the other types of cancer with 8p23 deletions. RT-PCR and EST data indicate that CSMD1 is also expressed in those organs,making this gene a candidate for a suppressor of multiple types of cancer. Both the sequence of the gene and the organization of the protein are highly conserved in the mouse.     

A novel giant gene CSMD3 encoding a protein with CUB and sushi multiple domains: a candidate gene for benign adult familial myoclonic epilepsy on human chromosome 8q23.3-q24.1

We identified a novel giant gene encoding a transmembrane protein with CUB and sushi multiple domains on the human chromosome 8q23.3-q24.1 in which benign adult familial myoclonic epilepsy type 1 (BAFME1/FAME, OMIM:601068) has been mapped. This giant gene consists of 73 exons and spans over 1.2Mb on the genomic DNA region. It showed significant homology to two genes, CSMD1 gene on 8p23 and CSMD2 gene on 1p34, at reduced amino acid sequence level and hence we designated as CSMD3. The CSMD3 gene was expressed mainly in adult and fetal brains. We performed mutation analysis on the CSMD3 gene for seven patients with BAFME1/FAME, but no mutation was found in the coding sequence of the CSMD3 gene. Comparative genomic analysis revealed a conserved family of CSMD genes in the mouse and fugu genomes. Possible functions of the CSMD gene family are discussed.     

The T cell differentiation antigen Leu-2/T8 is homologous to immunoglobulin and T cell receptor variable regions

Leu-2/T8 is a cell surface glycoprotein expressed by most cytotoxic and suppressor T lymphocytes. Its expression on T cells correlates best with recognition of class I major histocompatibility complex antigens, and it has been postulated to be a receptor for these proteins. We have determined the complete primary structure of Leu-2/T8 from the nucleotide sequence of its cDNA. The protein contains a classical signal peptide, two external domains, a hydrophobic transmembrane region, and a cytoplasmic tail. The N-terminal domain of the protein has striking homology to variable regions of immunoglobulins and the T cell receptor. The membrane-proximal domain appears to be a hinge-like region similar to that of immunoglobulin heavy chains. The superfamily of immunologically important surface molecules can now be extended to include Leu-2/T8.     

Janus kinases and focal adhesion kinases play in the 4.1 band: a superfamily of band 4.1 domains important for cell structure and signal transduction.

The band 4.1 domain was first identified in the red blood cell protein band 4.1, and subsequently in ezrin, radixin, and moesin (ERM proteins) and other proteins, including tumor suppressor merlin/schwannomin, talin, unconventional myosins VIIa and X, and protein tyrosine phosphatases. Recently, the presence of a structurally related domain has been demonstrated in the N-terminal region of two groups of tyrosine kinases: the focal adhesion kinases (FAK) and the Janus kinases (JAK). Additional proteins containing the 4.1/JEF (JAK, ERM, FAK) domain include plant kinesin-like calmodulin-binding proteins (KCBP) and a number of uncharacterized open reading frames identified by systematic DNA sequencing. Phylogenetic analysis of amino acid sequences suggests that band 4.1/JEF domains can be grouped in several families that have probably diverged early during evolution. Hydrophobic cluster analysis indicates that the band 4.1/JEF domains might consist of a duplicated module of approximately 140 residues and a central hinge region. A conserved property of the domain is its capacity to bind to the membrane-proximal region of the C-terminal cytoplasmic tail of proteins with a single transmembrane segment. Many proteins with band 4.1/JEF domains undergo regulated intra- or intermolecular homotypic interactions. Additional properties common to band 4.1/JEF domains of several proteins are binding of phosphoinositides and regulation by GTPases of the Rho family. Many proteins with band 4. 1/JEF domains are associated with the actin-based cytoskeleton and are enriched at points of contact with other cells or the extracellular matrix, from which they can exert control over cell growth. Thus, proteins with band 4.1/JEF domain are at the crossroads between cytoskeletal organization and signal transduction in multicellular organisms. Their importance is underlined by the variety of diseases that can result from their mutations.     

Mutants of the Rous sarcoma virus envelope glycoprotein that lack the transmembrane anchor and cytoplasmic domains: analysis of intracellular transport and assembly into virions.

The envelope glycoprotein complex of Rous sarcoma virus consists of a knoblike, receptor-binding gp85 polypeptide that is linked through disulfide bonds to a membrane-spanning gp37 spike. We used oligonucleotide-directed mutagenesis to assess the role of the hydrophobic transmembrane region and hydrophilic cytoplasmic domain of gp37 in intracellular transport and assembly into virions. Early termination codons were introduced on either side of the hydrophobic transmembrane region, and the mutated env genes were expressed from the late promoter of simian virus 40. This resulted in the synthesis of glycoprotein complexes composed of a normal gp85 and a truncated gp37 molecule that lacked the cytoplasmic domain alone or both the cytoplasmic and transmembrane domains. The biosynthesis and intracellular transport of the truncated proteins were not significantly different from those of the wild-type glycoproteins, suggesting that any protein signals for biosynthesis and intracellular transport of this viral glycoprotein complex must reside in its extracellular domain. The glycoprotein complex lacking the cytoplasmic domain of gp37 is stably expressed on the cell surface in a manner similar to that of the wild type. In contrast, the complex lacking both the transmembrane and cytoplasmic domains is secreted as a soluble molecule into the media. It can be concluded, therefore, that the transmembrane domain alone is essential for anchoring the RSV env complex in the cell membrane and that the cytoplasmic domain is not required for anchor function. Insertion of the mutated genes into an infectious proviral genome allowed us to assess the ability of the truncated gene products to be assembled into virions and to determine whether such virions were infectious. Viral genomes encoding the secreted glycoprotein were noninfectious, whereas those encoding a glycoprotein complex lacking only the cytoplasmic domain of gp37 were infectious. Virions produced from these mutant-infected cells contained normal levels of glycoprotein. The cytoplasmic tail of gp37 is thus not required for the assembly of envelope glycoproteins into virions. It is unlikely, therefore, that this region of gp37 interacts with viral core proteins during the selective incorporation of viral glycoproteins into the viral envelope.     

Genetic analysis of the cytoplasmic domain of the PfRh2b merozoite invasion protein of Plasmodium falciparum

Apicomplexan parasites employ multiple adhesive ligands for recognition and entry into host cells. The Duffy binding-like (DBL) and the reticulocyte binding protein-like (RBL) families are central to the invasion of erythrocytes by the malaria parasite. These type-1 transmembrane proteins are composed of large ectodomains and small conserved cytoplasmic tail domains. The cytoplasmic tail domain of the micronemal DBL protein EBA-175 is required for a functional ligand-receptor interaction, but not for correct trafficking and localisation. Here we focus on the cytoplasmic tail domain of the rhoptry-localised Plasmodium falciparum RBL PfRh2b. We have identified a conserved sequence of six amino acids, enriched in acidic residues, in the cytoplasmic tail domains of RBL proteins from Plasmodium spp. Genetic analyses reveal that the entire cytoplasmic tail and the conserved motif within the cytoplasmic tail are indispensable for invasion P. falciparum. Site-directed mutagenesis of the conserved moiety reveals that changes in the order of the amino acids of the conserved moiety, but not the charge of the sequence, can be tolerated. Shuffling of the motif has no effect on either invasion phenotype or PfRh2b expression and trafficking. Although the PfRh2b gene can be readily disrupted, our results suggest that modification of the PfRh2b cytoplasmic tail results in strong dominant negative activity, highlighting important differences between the PfRh2b and EBA-175 invasion ligands.     

Mutants of the membrane-binding region of Semliki Forest virus E2 protein. II. Topology and membrane binding

The p62/E2 protein of Semliki Forest virus (SFV) is a typical transmembrane glycoprotein, with an amino-terminal lumenal domain, a transmembrane (hydrophobic) domain, and a carboxy-terminal cytoplasmic domain (or tail). Our hypothesis has been that the membrane-binding polypeptide region (membrane anchor) of this protein consists of both the transmembrane domain and the adjacent positively charged peptide, Arg-Ser-Lys, which is part of the cytoplasmic domain. We have investigated three anchor mutants of the p62 protein with respect to both their disposition and their stability in cell membranes. The construction of the three mutants has been described (Cutler, D.F., and H. Garoff, J. Cell Biol., 102:889-901). They are as follows: A1, changing the basic charge cluster from Arg-Ser-Lys(+2) to Gly-Ser-Glu(-1); A2, replacing an Ala in the middle of the hydrophobic stretch with a Glu; A3, changing the charge cluster from Arg-Ser-Lys(+2) to Gly-Ser-Met(0). All three mutants retain the transmembrane configuration of the wild-type p62. In a cell homogenate they have a cytoplasmic domain that is accessible to protease. In living cells an anti-peptide antibody specific for the cytoplasmic tail of p62 reacts with the tails of both wild-type and mutant p62s following its introduction into the cytoplasm. All three mutant proteins have Triton X-114 binding properties similar to the wild-type p62. However, when the membranes of cells expressing the three mutants or the wild-type p62 protein are washed with sodium carbonate, pH 11.5, three to four times as much mutant protein as wild-type p62 is released from the membranes. Thus the stability in cell membranes of the three mutant p62 proteins is significantly reduced.     

Role of the alpha1 and alpha2 integrin cytoplasmic domains in cell morphology, motility and responsiveness to stimulation by the protein kinase C pathway

The alpha1beta1 and alpha2beta1 integrins, extracellular matrix receptors for collagens and/or laminins, have similarities in structure and ligand binding. Recent studies suggest that the two receptors mediate distinct post-ligand binding events and are not simply redundant receptors. To discern the mechanisms by which the two receptors differ, we focused on the roles of the cytoplasmic domains of the alpha subunits. We expressed either full-length alpha1 integrin subunit cDNA (X1C1), full-length alpha2 integrin subunit cDNA (X2C2), chimeric cDNA composed of the extracellular and transmembrane domains of alpha2 subunit and the cytoplasmic domain of alpha1 (X2C1), chimeric cDNA composed of the extracellular and transmembrane domains of alpha1 subunit and the cytoplasmic domain of alpha2 (X1C2), alpha1 cDNA truncated after the GFFKR sequence (X1C0) or alpha2 cDNA truncated after the GFFKR sequence (X2C0) in K562 cells. Although the cytoplasmic domains of the alpha1 and alpha2 subunits were not required for adhesion, the extent of adhesion at low substrate density was enhanced by the presence of either the alpha1 or alpha2 cytoplasmic tail. Spreading was also influenced by the presence of an alpha subunit cytoplasmic tail. Activation of the protein kinase C pathway with phorbol dibutyrate-stimulated motility that was dependent upon the presence of the alpha2 cytoplasmic tail. Both the phosphatidylinosotide-3-OH kinase and the mitogen-activated protein kinase pathways were required for phorbol-activated, alpha2-cytoplasmic tail-dependent migration.     

Oligomerization through hemopexin and cytoplasmic domains regulates the activity and turnover of membrane-type 1 matrix metalloproteinase.

The formation of multimeric complexes by membrane-type 1 matrix metalloproteinase (MT1-MMP) may facilitate its autocatalytic inactivation or proMMP-2 activation on the cell surface. To characterize these processes, we expressed various glutathione S-transferase/MT1-MMP fusion proteins in human HT-1080 fibrosarcoma cells and SV40-transformed lung fibroblasts and analyzed their effects on MT1-MMP activity and potential homophilic interactions. We report here that MT1-MMP is expressed on the cell surface as oligomeric 200--240-kDa complexes containing both the active 60-kDa and autocatalytically processed 43-kDa species. Overexpression of a glutathione S-transferase/MT1-MMP fusion protein containing the transmembrane and cytoplasmic domains of MT1-MMP inhibited the phorbol 12-myristate 13-acetate-induced autocatalytic cleavage of endogenous MT1-MMP to the 43-kDa species, but not proMMP-2 activation. On the other hand, a similar fusion protein with the hemopexin, transmembrane, and cytoplasmic domains inhibited proMMP-2 activation in a dominant-negative fashion. These results suggest that both the autocatalytic cleavage of MT1-MMP and proMMP-2 activation may be regulated by oligomerization through the cytoplasmic and hemopexin domains. Indeed, either domain, when attached to the cell membrane by a transmembrane domain, formed stable homophilic complexes. Copurification of MT1-MMP with these fusion proteins correlated with their cell-surface co-localization. Thus, MT1-MMP oligomerization through the hemopexin, transmembrane, and cytoplasmic domains controls its catalytic activity.     

Protein interaction analysis of ST14 domains and their point and deletion mutants

ST14 (suppression of tumorigenicity 14) is a transmembrane serine protease that contains a serine protease catalytic (SP) domain, an SEA domain, two complement subcomponent C1r/s (CUB) domains, and four low density lipoprotein receptor class A domains. Glutathione S-transferase fusion proteins with SP, CUB, and low density lipoprotein receptor domains and their corresponding mutants were generated to analyze protein interactions with these domains. Modified glutathione S-transferase pull-down assays demonstrated the interaction between the SP domain and hepatocyte growth factor activator inhibitor-1. With the same method, a CUB domain-interacting protein was isolated and turned out to be the transmembrane protein with epidermal growth factor-like and two follistatin-like domains 1 (TMEFF1). Quantitative real time PCR revealed that the expression of the TMEFF1 gene was dependent on the transfection of the ST14 gene in the RKO cell line. Our results also suggested that ST14 and TMEFF1 were co-expressed in the human breast cancer cell line MCF7, human placenta, kidney, and liver tissues. Interestingly, these two genes were co-up-regulated in kidney tumors versus normal tissues, consistent with our results that showed the dependence of TMEFF1 expression on ST14 in RKO cells. Finally, homology modeling studies suggested that TMEFF1 might form a complex with ST14 by an interaction between epidermal growth factor and CUB domains.     

Domain and functional analysis of a novel breast tumor suppressor protein, SCUBE2

Signal peptide CUB (complement proteins C1r/C1s, Uegf, and Bmp 1)-EGF domain-containing protein 2 (SCUBE2) is a secreted, membrane-associated multidomain protein composed of five recognizable motifs: an NH(2)-terminal signal peptide sequence, nine copies of epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeats, and one CUB domain at the COOH terminus. Our previous clinical study showed that SCUBE2 may act as a novel breast tumor suppressor gene and serve as a useful prognostic marker. However, the specific domain responsible for its tumor suppressor activity and the precise mechanisms of its anti-tumor effect remain unknown. Using a combination of biochemical, molecular, and cell biology techniques, we further dissected the molecular functions and signal pathways mediated by the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain of SCUBE2. Independent overexpression of the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain resulted in suppression of MCF-7 breast cancer cell proliferation and reduced MCF-7 xenograft tumor growth in nude mice. Molecular and biochemical analyses revealed that the COOH-terminal CUB domain could directly bind to and antagonize bone morphogenetic protein activity in an autocrine manner, whereas the NH(2)-terminal EGF-like repeats could mediate cell-cell homophilic adhesions in a calcium-dependent fashion, interact with E-cadherin (a master tumor suppressor), and decrease the β-catenin signaling pathway. Together, our data demonstrate that SCUBE2 has growth inhibitory effects through a coordinated regulation of two distinct mechanisms: antagonizing bone morphogenetic protein and suppressing the β-catenin pathway in breast cancer cells.     

Structure and function of integral membrane protein domains resolved by peptide-amphiphiles: application to phospholamban

We have used synthetic lipidated peptides ("peptide-amphiphiles") to study the structure and function of isolated domains of integral transmembrane proteins. We used 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis to prepare full-length phospholamban (PLB(1-52)) and its cytoplasmic (PLB(1-25)K: phospholamban residues 1-25 plus a C-terminal lysine), and transmembrane (PLB(26-52)) domains, and a 38-residue model alpha-helical sequence as a control. We created peptide-amphiphiles by linking the C-terminus of either the isolated cytoplasmic domain or the model peptide to a membrane-anchoring, lipid-like hydrocarbon tail. Circular dichroism measurements showed that the model peptide-amphiphile, either in aqueous suspension or in lipid bilayers, had a higher degree of alpha-helical secondary structure than the unlipidated model peptide. We hypothesized that the peptide-amphiphile system would allow us to study the function and structure of the PLB(1-25)K cytoplasmic domain in a native-like configuration. We compared the function (inhibition of the Ca-ATPase in reconstituted membranes) and structure (via CD) of the PLB(1-25) amphiphile to that of PLB and its isolated transmembrane and cytoplasmic domains. Our results indicate that the cytoplasmic domain PLB(1-25)K has no effect on Ca-ATPase (calcium pump) activity, even when tethered to the membrane in a manner mimicking its native configuration, and that the transmembrane domain of PLB is sufficient for inhibition of the Ca-ATPase.     

The transmembrane domain of murine alpha-mannosidase IB is a major determinant of Golgi localization

Murine alpha1,2-mannosidase IB is a type II transmembrane protein localized to the Golgi apparatus where it is involved in the biogenesis of complex and hybrid N-glycans. This enzyme consists of a cytoplasmic tail, a transmembrane domain followed by a "stem" region and a large C-terminal catalytic domain. To analyze the determinants of targeting, we constructed various deletion mutants of murine alpha1,2-mannosidase IB as well as alpha1,2-mannosidase IB/yeast alpha1,2-mannosidase and alpha1,2-mannosidase IB/GFP chimeras and localized these proteins by fluorescence microscopy, when expressed transiently in COS7 cells. Replacing the catalytic domain of alpha1,2-mannosidase IB with that of the homologous yeast alpha1,2-mannosidase and deleting the "stem" region in this chimera had no effect on Golgi targeting, but caused increased cell surface localization. The N-terminal tagged protein lacking a catalytic domain was also localized to the Golgi. In the latter case, when the stem region was partially or completely removed, the protein was found in both the ER and the Golgi. A chimera consisting of the alpha1,2-mannosidase IB N-terminal region (cytoplasmic and transmembrane domains plus 10 amino acids of the "stem" region) and GFP was localized mainly to the Golgi. Deletion of 30 out of 35 amino acids in the cytoplasmic tail had no effect on Golgi localization. A GFP chimera lacking the entire cytoplasmic tail was found in both the ER and the Golgi. These results indicate that the transmembrane domain of alpha1,2-mannosidase IB is a major determinant of Golgi localization.     

CSMD1 exhibits antitumor activity in A375 melanoma cells through activation of the Smad pathway

In this work, we studied the effects of CUB and Sushi multiple domains 1 gene (CSMD1) expression in A375 melanoma cells in vivo and in vitro. CSDM1 expression decreased proliferation and migration, and increased apoptosis and G(1) arrest in A375 cells in vitro. Expression of CSDM1 in established xenografted tumors decreased tumor size and weight, and decreased the density of intratumor microvessels. The survival rate of mice with tumors expressing CSMD1 was significantly higher than mice with tumors that did not express CSDM1. These results confirm the role of CSDM1 as a tumor suppressor gene in melanoma cells. Furthermore, we found that CSMD1 can interact with Smad3, activate Smad1, Smad2, and Smad3, and increase the expression of Smad4. These results might prove helpful for the development of novel therapies for melanoma treatment.     

The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase.

The CLAVATA2 (CLV2) gene regulates both meristem and organ development in Arabidopsis. We isolated the CLV2 gene and found that it encodes a receptor-like protein (RLP), with a presumed extracellular domain composed of leucine-rich repeats similar to those found in plant and animal receptors, but with a very short predicted cytoplasmic tail. RLPs lacking cytoplasmic signaling domains have not been previously shown to regulate development in plants. Our prior work has demonstrated that the CLV1 receptor-like kinase (RLK) is present as a disulfide-linked multimer in vivo. We report that CLV2 is required for the normal accumulation of CLV1 protein and its assembly into protein complexes, indicating that CLV2 may form a heterodimer with CLV1 to transduce extracellular signals. Sequence analysis suggests that the charged residue in the predicted transmembrane domain of CLV2 may be a common feature of plant RLPs and RLKs. In addition, the chromosomal region in which CLV2 is located contains an extremely high rate of polymorphism, with 50 nucleotide and 15 amino acid differences between Landsberg erecta and Columbia ecotypes within the CLV2 coding sequence.     

A specific transmembrane domain of a coronavirus E1 glycoprotein is required for its retention in the Golgi region

The E1 glycoprotein of the avian coronavirus infectious bronchitis virus contains a short, glycosylated amino-terminal domain, three membrane-spanning domains, and a long carboxy-terminal cytoplasmic domain. We show that E1 expressed from cDNA is targeted to the Golgi region, as it is in infected cells. E1 proteins with precise deletions of the first and second or the second and third membrane-spanning domains were glycosylated, thus suggesting that either the first or third transmembrane domain can function as an internal signal sequence. The mutant protein with only the first transmembrane domain accumulated intracellularly like the wild-type protein, but the mutant protein with only the third transmembrane domain was transported to the cell surface. This result suggests that information specifying accumulation in the Golgi region resides in the first transmembrane domain, and provides the first example of an intracellular membrane protein that is transported to the plasma membrane after deletion of a specific domain.     

The cytoplasmic tail of alpha 1,3-galactosyltransferase inhibits Golgi localization of the full-length enzyme.

It is currently under debate whether the mechanism of Golgi retention of different glycosyltransferases is determined by sequences in the transmembrane, luminal, or cytoplasmic domains or a combination of these domains. We have shown that the cytoplasmic domains of alpha1,3-galactosyltransferase (GT) and alpha1,2-fucosyltransferase (FT) are involved in Golgi localization. Here we show that the cytoplasmic tails of GT and FT are sufficient to confer specific Golgi localization. Further, we show that the expression of only the cytoplasmic tail of GT can lead to displacement or inhibition of binding of the whole transferase and that cells expressing the cytoplasmic tail of GT were not able to express full-length GT or its product, Galalpha1,3Gal. Thus, the presence of the cytoplasmic tail prevented the localization and function of full-length GT, suggesting a possible specific Golgi binding site for GT. The effect was not altered by the inclusion of the transmembrane domain. Although the transmembrane domain may act as an anchor, these data show that, for GT, only the cytoplasmic tail is involved in specific localization to the Golgi.     

Structural Insights into Calmodulin-regulated L-selectin Ectodomain Shedding

The L-selectin glycoprotein receptor mediates the initial steps of leukocyte migration into secondary lymphoid organs and sites of inflammation. Following cell activation through the engagement of G-protein-coupled receptors or immunoreceptors, the extracellular domains of L-selectin are rapidly shed, a process negatively controlled via the binding of the ubiquitous eukaryotic calcium-binding protein calmodulin to the cytoplasmic tail of L-selectin. Here we present the solution structure of calcium-calmodulin bound to a peptide encompassing the cytoplasmic tail and part of the transmembrane domain of L-selectin. The structure and accompanying biophysical study highlight the importance of both calcium and the transmembrane segment of L-selectin in the interaction between these two proteins, suggesting that by binding this region, calmodulin regulates in an "inside-out" fashion the ectodomain shedding of the receptor. Our structure provides the first molecular insight into the emerging new role for calmodulin as a transmembrane signaling partner.