Putative Cell Surface Receptor Research Articles

LilrB3 is a putative cell surface receptor of APOE4.

The three isoforms of apolipoprotein E (APOE2, APOE3, and APOE4) only differ in two amino acid positions but exert quite different immunomodulatory effects. The underlying mechanism of such APOE isoform dependence remains enigmatic. Here we demonstrate that APOE4, but not APOE2, specifically interacts with the leukocyte immunoglobulin-like receptor B3 (LilrB3). Two discrete immunoglobin-like domains of the LilrB3 extracellular domain (ECD) recognize a positively charged surface patch on the N-terminal domain (NTD) of APOE4. The atomic structure reveals how two APOE4 molecules specifically engage two LilrB3 molecules, bringing their intracellular signaling motifs into close proximity through formation of a hetero-tetrameric complex. Consistent with our biochemical and structural analyses, APOE4, but not APOE2, activates human microglia cells (HMC3) into a pro-inflammatory state in a LilrB3-dependent manner. Together, our study identifies LilrB3 as a putative immune cell surface receptor for APOE4, but not APOE2, and may have implications for understanding the biological functions as well as disease relevance of the APOE isoforms.

KDEL receptor is a cell surface receptor that cycles between the plasma membrane and the Golgi via clathrin-mediated transport carriers.

KDEL receptor cycles between the ER and the Golgi to retrieve ER-resident chaperones that get leaked to the secretory pathway during protein export from the ER. Recent studies have shown that a fraction of KDEL receptor may reside in the plasma membrane and function as a putative cell surface receptor. However, the trafficking itinerary and mechanism of cell surface expressed KDEL receptor remains largely unknown. In this study, we used N-terminally Halo-tagged KDEL receptor to investigate its endocytosis from the plasma membrane and trafficking itinerary of the endocytosed receptor through the endolysosomal compartments. Our results indicate that surface-expressed KDEL receptor undergoes highly complex recycling pathways via the Golgi and peri-nuclear recycling endosomes that are positive for Rab11 and Rab14, respectively. Unexpectedly, KDEL receptor appears to preferentially utilize clathrin-mediated endocytic pathway as well as clathrin-dependent transport carriers for export from the trans-Golgi network. Taken together, we suggest that KDEL receptor may be a bona fide cell surface receptor with a complex, yet well-defined trafficking itinerary through the endolysosomal compartments.

Distinct Contributions of CD18 Integrins for Binding and Phagocytic Internalization of Pseudomonas aeruginosa

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.

A CD133-AKT-Wnt signaling axis drives glioblastoma brain tumor-initiating cells.

Mechanistic insight into signaling pathways downstream of surface receptors has been revolutionized with integrated cancer genomics. This has fostered current treatment modalities, namely immunotherapy, to capitalize on targeting key oncogenic signaling nodes downstream of a limited number of surface markers. Unfortunately, rudimentary mechanistic understanding of most other cell surface proteins has reduced the clinical utility of these markers. CD133 has reproducibly been shown to correlate with disease progression, recurrence, and poor overall survivorship in the malignant adult brain tumor, glioblastoma (GBM). Using several patient-derived CD133high and CD133low GBMs we describe intrinsic differences in determinants of stemness, which we owe to a CD133-AKT-Wnt signaling axis in which CD133 functions as a putative cell surface receptor for AKT-dependent Wnt activation. These findings may have implications for personalized oncology trials targeting PI3K/AKT or Wnt as both pathways may be activated independent of their canonical drivers, leading to treatment resistance and disease relapse.

Caged ceramide 1-phosphate (C1P) analogs: Novel tools for studying C1P biology

Ceramide 1-phosphate (C1P) is a bioactive sphingolipid metabolite that is produced in cells by the action of ceramide kinase (CerK) acting upon ceramide, and is also found in the circulation. C1P was first demonstrated to be mitogenic and antiapoptotic in different cell types, and was later shown to induce cell migration. Understanding the precise mechanisms by which C1P exerts its biological effects has been possible using specific photosensitive caged C1P analogues synthesized by Robert Bittman’s group. These compounds are cell permeable, bypass cell plasma membrane receptors, and can be released into the cytosol upon light irradiation, thereby allowing precise determination of the intracellular mechanisms of actions of C1P. Two derivatives of N-palmitoyl-ceramide 1-phosphate have been used in most studies. In one C1P derivative the cage was 7-(N,N-diethylamino)coumarin (DECM-C1P) while in the other it was a 4-bromo-5-hydroxy-2-nitrobenzhydryl moiety (BHNB-C1P). The uncaging process released C1P in the cytosol, and this was accompanied by stimulation of cell proliferation, inhibition of apoptosis, and production of low levels of reactive oxygen species. However, intracellular accumulation of C1P did not affect chemotaxis. The caged C1P analogues allowed distinction between the extracellular events evoked by C1P, as for example through interaction with a putative cell-surface receptor, from its intracellular effects.

An alternative receptor to poly I:C on cell surfaces for interferon induction

Not-self or denatured nucleic acids are recognized by pattern recognition receptors localized mainly in endosomes and cytoplasm, such as Toll-like receptor (TLR) 3, TLR7, TLR9, retinoic acid-inducible gene-I, DNA-dependent activator of IFN-regulatory factors and other receptors. The binding of polyriboinosinic:polyribocytidylic acid (poly I:C), a synthetic dsRNA that robustly induces type I interferon, to a putative cell-surface receptor on a rabbit kidney cell line, RK13, has been analyzed by the authors and RK13 cells found to capture poly I:C in a specific fashion with sufficient affinity. These findings suggest that an alternative receptor to poly I:C participates in the induction of type 1 interferon, which localizes on cell surfaces. Although the nature of this molecule has not yet been identified, accumulating evidence has led the present authors to speculate that there are undefined classes of RNA-recognition molecules on cell surfaces and that these are unlikely to be categorized as previously reported dsRNA receptors. Although many years have passed since this possibility was first reported by the present authors, it remains attractive. In this article, previously reported cell-surface dsRNA receptors are reviewed in comparison with other receptors reported to date that are firmly involved in the innate immune-sensing of nucleic acids.

An alternative receptor to Poly I:C on cell surface for interferon induction

Not-self or denatured nucleic acids are recognized by pattern recognition receptors localized mainly in endosomes and cytoplasm, such as Toll-like receptor (TLR) 3, TLR7, TLR9, retinoic acid-inducible gene-I, DNA-dependent activator of IFN-regulatory factors and other receptors. The binding of polyriboinosinic:polyribocytidylic acid (poly I:C), a synthetic dsRNA that robustly induces type I interferon, to a putative cell-surface receptor on a rabbit kidney cell line, RK13, has been analyzed by the authors and RK13 cells found to capture poly I:C in a specific fashion with sufficient affinity. These findings suggest that an alternative receptor to poly I:C participates in the induction of type 1 interferon, which localizes on cell surfaces. Although the nature of this molecule has not yet been identified, accumulating evidence has led the present authors to speculate that there are undefined classes of RNA-recognition molecules on cell surfaces and that these are unlikely to be categorized as previously reported dsRNA receptors. Although many years have passed since this possibility was first reported by the present authors, it remains attractive. In this article, previously reported cell-surface dsRNA receptors are reviewed in comparison with other receptors reported to date that are firmly involved in the innate immune-sensing of nucleic acids.

A Putative Cell Surface Receptor for White Spot Syndrome Virus Is a Member of a Transporter Superfamily

White spot syndrome virus (WSSV), a large enveloped DNA virus, can cause the most serious viral disease in shrimp and has a wide host range among crustaceans. In this study, we identified a surface protein, named glucose transporter 1 (Glut1), which could also interact with WSSV envelope protein, VP53A. Sequence analysis revealed that Glut1 is a member of a large superfamily of transporters and that it is most closely related to evolutionary branches of this superfamily, branches that function to transport this sugar. Tissue tropism analysis showed that Glut1 was constitutive and highly expressed in almost all organs. Glut1's localization in shrimp cells was further verified and so was its interaction with Penaeus monodon chitin-binding protein (PmCBP), which was itself identified to interact with an envelope protein complex formed by 11 WSSV envelope proteins. In vitro and in vivo neutralization experiments using synthetic peptide contained WSSV binding domain (WBD) showed that the WBD peptide could inhibit WSSV infection in primary cultured hemocytes and delay the mortality in shrimps challenged with WSSV. These findings have important implications for our understanding of WSSV entry.

Review of the putative cell-surface receptors for alpha-fetoprotein: identification of a candidate receptor protein family

The identification of a receptor for alpha-fetoprotein (AFP) has long been sought in the field of medicine. The uptake and endocytosis of AFP by rat tumor cells in 1984 sparked a series of confirmatory reports and the original studies were then extended to include multiple tumor types in rats, mice, and humans. The following year, French investigators partially characterized the binding properties of the AFP receptor, but they were not able to purify the receptor. It was not until 1991-1992 that an AFP receptor was partially purified and characterized from both human monocytes and breast cancer cells. By 1993, a monoclonal antibody had been raised against the AFP receptor produced from a breast cancer extract with claims that the receptor was a widespread (universal) oncofetal biomarker for cancer. However, that receptor has yet to be cloned and/or purified due to its complex multimeric binding interactions and associations. The present report will review the literature of the multiple putative AFP receptors described to date, the cellular uptake and endocytosis of AFP, and the biochemical characterization of these putative cell-surface proteins. In addition, evidence derived from computer modeling, proteolytic degradation patterns, and amino acid sequence analysis will be presented in a proposed identification of a family of multi-ligand binding receptors; this family fits many, if not most, of the criteria required for an AFP receptor. The purposed receptor protein family is tentatively identified as the Scavenger receptors which comprise several classes of single- and double-pass integral transmembrane proteins. Present data do not support the concept that the AFP receptor is a "universal" tumor receptor and/or biomarker.

Structure–activity studies of GmSubPep, a soybean peptide defense signal derived from an extracellular protease

GmSubPep, a 12-amino acid peptide isolated from soybean leaves, induces the expression of genes in soybean suspension-cultured cells that encode proteins involved in defense against pathogens. The peptide is derived from an extracellular subtilisin-like protease (subtilase) and binds a putative cell-surface receptor that initiates a defense signaling cascade. Interaction of the peptide with its receptor results in alkalinization of soybean suspension cell media which can be utilized to analyze the kinetics of receptor binding. Substitutions of alanine at each of the 12 amino acid positions revealed that the amino acids at positions 10 (arginine) and 12 (histidine) were essential for activity. Both analogs were able to reduce the physiological effects of GmSubPep associated with receptor binding. Deletion of the C-terminal histidine [GmSubPep(1–11)] abolished the alkalinizing activity and this peptide was also a strong competitor for receptor binding. Deletion of N-terminal amino acids from GmSubPep caused a sequential loss of activity with no alkalinizing activity for GmSubPep(4–12). However, the N-terminal deleted peptides did not compete with GmSubPep for receptor binding. Further modifications at the arginine-10 position indicated that an ionizable proton was not essential for activity as an attenuated response was found for a citrulline substitution. Substitution of the histidine-12 with methylated histidine at position N-1 of the imidazole group abolished activity, whereas substitution at N-3 was completely active, indicating that the N-3 analog retains important receptor binding properties. This study indicates that the extreme C-terminal of GmSubPep has important signal transduction properties while the C-terminal is essential for receptor interaction.

Molecular Characterization of the Full-Length Coding Sequence of the Caprine Laminin Receptor Gene (RPSA)

Scrapie is a prion disease in sheep and goats. Ribosomal protein SA (RPSA), also called 37 kDa laminin receptor precursor/67 kDa laminin receptor has been demonstrated to be a putative cell surface receptor for prion. To investigate the caprine RPSA, we cloned the full-length coding sequence of the gene of goat and submitted it to GenBank. The length of the open reading frame is 888 bp, encoding 295 amino acids. The putative amino acid sequence is highly similar to that of other mammals. The caprine amino acid sequence of RPSA is shown to be identical to the sequence of species susceptible to scrapie at positions 241, 272, and 291. The phylogenetic tree analysis revealed that the genetic distance between sheep and goat is the smallest. Moreover, RT-PCR results of 11 tissues indicated that RPSA mRNA is expressed in all selected caprine tissues.

Effects of Specific Genes Activating RAGE on Polycystic Kidney Disease

Background: Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by progressive cyst formation and secretion of fluid and is associated with interstitial inflammation and fibrosis, resulting in the loss of renal function. We previously generated mice overexpressing PKD2, causing progressive cyst development with an inflammatory and fibrotic phenotype in the kidneys. Methods: To profile the gene expression related to inflammation and cystogenesis, microarray analysis was performed with kidney tissue from 6-, 12- and 18-month-old mice. Subsequently, levels and related mechanisms of selected genes, s100a8 and s100a9, were evaluated. Results: S100a8 and s100a9 was upregulated more than 2-fold and differently expressed in the cystic region. Receptor of advanced glycation end product (RAGE) is a putative cell surface receptor for s100a8/a9. It was expressed in cyst-lining cells and up-regulated pro-inflammatory transcription factor NF-ĸB in transgenic mice. We also confirmed RAGE expression in ADPKD patient kidneys. It was suggested that the signaling related to proliferative cystogenesis through previous reports; therefore, we confirmed that phosphorylated-ERK and cyst formation was reduced by treatment of RAGE-siRNA. Conclusions: The results may provide important information for the expression of s100a8/a9 and RAGE, linking progressive cystogenesis with inflammation in cystic kidney.

The apical transmembrane protein Crumbs functions as a tumor suppressor that regulates Hippo signaling by binding to Expanded

The Hippo signaling pathway regulates organ size and tissue homeostasis from Drosophila to mammals. At the core of the Hippo pathway is a kinase cascade extending from the Hippo (Hpo) tumor suppressor to the Yorkie (Yki) oncoprotein. The Hippo kinase cascade, in turn, is regulated by apical membrane-associated proteins such as the FERM domain proteins Merlin and Expanded (Ex), and the WW- and C2-domain protein Kibra. How these apical proteins are themselves regulated remains poorly understood. Here, we identify the transmembrane protein Crumbs (Crb), a determinant of epithelial apical-basal polarity in Drosophila embryos, as an upstream component of the Hippo pathway in imaginal disk growth control. Loss of Crb leads to tissue overgrowth and target gene expression characteristic of defective Hippo signaling. Crb directly binds to Ex through its juxtamembrane FERM-binding motif (FBM). Loss of Crb or mutation of its FBM leads to mislocalization of Ex to basolateral domain of imaginal disk epithelial cells. These results shed light on the mechanism of Ex regulation and provide a molecular link between apical-basal polarity and tissue growth. Furthermore, our studies implicate Crb as a putative cell surface receptor for Hippo signaling by uncovering a transmembrane protein that directly binds to an apical component of the Hippo pathway.

Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: Role of macrophage scavenger receptor class A type I and II

The Maillard reaction occurs between reducing sugars and proteins during thermal processing of foods. It produces chemically glycated proteins termed advanced glycation end products (AGEs). The glycation structures of AGEs are suggested to function as pathogenesis-related immune epitopes in food allergy. This study aimed at defining the T-cell immunogenicity of food AGEs by using ovalbumin (OVA) as a model allergen. AGE-OVA was prepared by means of thermal processing of OVA in the presence of glucose. Activation of OVA-specific CD4(+) T cells by AGE-OVA was evaluated in cocultures with bone marrow-derived murine myeloid dendritic cells (mDCs) as antigen-presenting cells. The uptake mechanisms of mDCs for AGE-OVA were investigated by using inhibitors of putative cell-surface receptors for AGEs, as well as mDCs deficient for these receptors. Compared with the controls (native OVA and OVA thermally processed without glucose), AGE-OVA enhanced the activation of OVA-specific CD4(+) T cells on coculture with mDCs, indicating that the glycation of OVA enhanced the T-cell immunogenicity of the allergen. The mDC uptake of AGE-OVA was significantly higher than that of the controls. We identified scavenger receptor class A type I and II (SR-AI/II) as a mediator of the AGE-OVA uptake, whereas the receptor for AGEs and galectin-3 were not responsible. Importantly, the activation of OVA-specific CD4(+) T cells by AGE-OVA was attenuated on coculture with SR-AI/II-deficient mDCs. SR-AI/II targets AGE-OVA to the MHC class II loading pathway in mDCs, leading to an enhanced CD4(+) T-cell activation. The Maillard reaction might thus play an important role in the T-cell immunogenicity of food allergens.

Cloning and characterization of full-length coding sequence (CDS) of the ovine 37/67-kDa laminin receptor (RPSA)

The 37-kDa Laminin Receptor Precursor (LRP)/67-kDa Laminin Receptor (LR), also known as ribosomal protein SA (RPSA), had been identified as a putative cell surface receptor for prions. Herein, we isolated the full-length coding sequence (CDS) of the ovine 37/67-kDa LRP/LR gene and submitted it to the GenBank under accession number EF649775. The open reading frame (ORF) of the 37/67-kDa LRP/LR CDS is 885 bp in length, containing six exons encoding a protein of 295 amino acids. The nucleotide sequence presented here is well coincided with the whole ovine genome of the 37/67-kDa LRP/LR previously published. Moreover, we identified four novel single nucleotide polymorphism sites (SNPs) at position 324 in exon 4, positions at 809, 875, and 881 in exon 7, respectively. Further, based on the deduced amino acid sequence alignment of the 37/67-kDa LRP/LR from human, cattle, mice, pig, chicken, and sheep, we also identified three polymorphic amino acid sites (PAAs) at residues 241, 272, and a novel site at residue 270 in the putative indirect prion protein (PrP) interaction region (180-285) on 37/67-kDa LRP/LR. Prediction of protein secondary structure further indicated that PAAs at residues 241, 270 and 272 may cause protein conformation changes as predicted, which may affect on the binding with prion protein. In addition, multiple-tissues RT-PCR results revealed that 37/67-kDa LRP/LR mRNA is expressed in all the 11 selected ovine tissues.

A Peptide Derived from Tenascin-C Induces β1 Integrin Activation through Syndecan-4

Tenascin-C (TN-C) is unique for its cell adhesion modulatory function. We have shown that TNIIIA2, a synthetic 22-mer peptide derived from TN-C, stimulated beta1 integrin-mediated cell adhesion of nonadherent and adherent cell types, by inducing activation of beta1 integrin. The active site of TNIIIA2 appeared cryptic in the TN-C molecule but was exposed by MMP-2 processing of TN-C. The following results suggest that cell surface heparan sulfate (HS) proteoglycan (HSPG), including syndecan-4, participated in TNIIIA2-induced beta1 integrin activation: 1) TNIIIA2 bound to cell surface HSPG via its HS chains, as examined by photoaffinity labeling; 2) heparitinase I treatment of cells abrogated beta1 integrin activation induced by TNIIIA2; 3) syndecan-4 was isolated by affinity chromatography using TNIIIA2-immobilized beads; 4) small interfering RNA-based down-regulation of syndecan-4 expression reduced TNIIIA2-induced beta1 integrin activation, and consequent cell adhesion to fibronectin; 5) overexpression of syndecan-4 core protein enhanced TNIIIA2-induced activation of beta1 integrin. However, treatments that targeted the cytoplasmic region of syndecan-4, including ectopic expression of its mutant truncated with the cytoplasmic domains and treatment with protein kinase Calpha inhibitor Gö6976, did not influence the TNIIIA2 activity. These results suggest that a TNIIIA2-related matricryptic site of the TN-C molecule, exposed by MMP-2 processing, may have bound to syndecan-4 via its HS chains and then induced conformational change in beta1 integrin necessary for its functional activation. A lateral interaction of beta1 integrin with the extracellular region of the syndecan-4 molecule may be involved in this conformation change.

Nicotinic acetylcholine receptor structure and function in the efferent auditory system

This article reviews and presents new data regarding the nicotinic acetylcholine receptor subunits alpha9 and alpha10. Although phylogentically ancient, these subunits have only recently been identified as critical components of the efferent auditory system and medial olivocochlear pathway. This pathway is important in auditory processing by modulating outer hair cell function to broadly tune the cochlea and improve signal detection in noise. Pharmacologic properties of the functionally expressed alpha9alpha10 receptor closely resemble the cholinergic response of outer hair cells. Molecular, immunohistochemical, and knockout mice studies have added further weight to the role this receptor plays in mediating the efferent auditory response. Alternate and complementary mechanisms of outer hair cell efferent activity might also be mediated through the nAChR alpha9alpha10, either through secondary calcium stores, second messengers, or direct protein-protein interactions. We investigated protein-protein interactions using a yeast-two-hybrid screen of the nAChR alpha10 intracellular loop against a rat cochlear cDNA library. Among the identified proteins was prosaposin, a precursor of saposins, which have been shown to act as neurotrophic factors in culture, can bind to a putative G0-coupled cell surface receptor, and may be involved in the prevention of cell death. This study and review suggest that nAChR alpha9alpha10 may represent a potential therapeutic target for a variety of ear disorders, including preventing or treating noise-induced hearing loss, or such debilitating disorders as vertigo or tinnitus.

Elucidation of N-Glycosylation Sites on Human Platelet Proteins

Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets.

FLRT3 is expressed in sensory neurons after peripheral nerve injury and regulates neurite outgrowth

We used a molecular screen to identify genes upregulated in regenerating adult rat dorsal root ganglion cells. FLRT3 mRNA and protein characterized by a fibronectin type III domain and a leucine-rich repeat motif was upregulated in damaged sensory neurons. The protein was then transported into their peripheral and central processes where the FLRT3 protein was localized to presynaptic axon terminals. In vitro, the FLRT3 protein was expressed at the cell surface, regulated neurite outgrowth in sensory neurons, but did not exhibit homophilic binding. FLRT3 was widely expressed in the developing embryo, particularly in the central nervous system and somites. However, in the adult, we found no evidence for accumulation or reexpression of the FLRT3 protein in damaged axons of the central nervous system. We conclude that FLRT3 codes for a putative cell surface receptor implicated in both the development of the nervous system and in the regeneration of the peripheral nervous system (PNS).

Annexins expressed on the cell surface serve as receptors for adhesion to immobilized fetuin-A

Fetuin-A is a major constituent of the fetal bovine serum used extensively in cell culture media. We hereby present data demonstrating that breast carcinoma cells can adhere to immobilized fetuin-A in a calcium-dependent fashion. Interestingly, the cells can also divide and attain confluency under these conditions. Using a proteomic approach, we have identified annexin-II and -VI as the putative cell surface receptors for fetuin-A in the presence of Ca 2+ ions. Biotinylation of cell surface proteins followed by immunoprecipitation revealed that annexin-VI was expressed on the extracytoplasmic surface of the cell membranes. Finally, to demonstrate that annexin-II and -VI were the adhesive receptors for fetuin-A, siRNA knockdown of expression of the annexins significantly reduced the calcium-mediated adhesion. Interestingly, we demonstrated that the tumor cells could also adhere to immobilized fetuin-A in the presence of magnesium ions, and that this adhesion was most likely mediated by integrins because neutralizing antibodies against β1 integrins substantially reduced the adhesion. Our studies suggest that the expression of annexin-II and -VI and possibly other members of the family mediate novel adhesion and signaling mechanisms in tumor cells.