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Monoclonal antibodies (mAb) and natural ligands targeting costimulatory tumor necrosis factor receptors (TNFR) exhibit a wide range of agonistic activities and antitumor responses. The mechanisms underlying these differential agonistic activities remain poorly understood. Here, we employ a panel of experimental and clinically-relevant molecules targeting human CD40, 4-1BB and OX40 to examine this issue. Confocal and STORM microscopy reveal that strongly agonistic reagents induce clusters characterized by small area and high receptor density. Using antibody pairs differing only in isotype we show that hIgG2 confers significantly more receptor clustering than hIgG1 across all three receptors, explaining its greater agonistic activity, with receptor clustering shielding the receptor-agonist complex from further molecular access. Nevertheless, discrete receptor clustering patterns are observed with different hIgG2 mAb, with a unique rod-shaped assembly observed with the most agonistic mAb. These findings dispel the notion that larger receptor clusters elicit greater agonism, and instead point to receptor density and subsequent super-structure as key determinants., Yu et al examined a panel of both experimental and clinically-relevant TNF agonists in order to advance our understanding of the mechanisms underlying their varying activities and anti-tumor responses. They demonstrated that agonists with greater activity induced higher density receptor clustering and specific super-structures as opposed to simply larger receptor clusters.

Lysine-specific demethylase 1 (LSD1/KDM1A) oxidatively removes methyl groups from histone proteins, and its aberrant activity has been correlated with cancers including acute myeloid leukemia (AML). We report a novel series of tranylcypromine analogues with a carboxamide at the 4-position of the aryl ring. These compounds, such as 5 a and 5 b with benzyl and phenethylamide substituents, respectively, had potent sub-micromolar IC 50 values for the inhibition of LSD1 as well as cell proliferation in a panel of AML cell lines. The dose-dependent increase in cellular expression levels of H3K4me2, CD86, CD11b and CD14 supported a mechanism involving LSD1 inhibition. The tert-butyl and ethyl carbamate derivatives of these tranylcypromines, although inactive in LSD1 inhibition, were of similar potency in cell-based assays with a more rapid onset of action. This suggests that carbamates can act as metabolically labile tranylcypromine prodrugs with superior pharmacokinetics.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2 process N-terminally extended antigenic precursors for optimal loading onto major histocompatibility complex class I (MHC I) molecules. We and others have demonstrated that ERAP1 processes peptides bound to MHC I, but the underlying mechanism is unknown. To this end, we utilized single-chain trimers (SCT) of the ovalbumin-derived epitope SIINFEKL (SL8) tethered to the H2-Kb MHC I determinant from mouse and introduced three substitutions, E63A, K66A, and W167A, at the A-pocket of the peptide-binding groove in the MHC I heavy chain, which interact with the N termini of peptides. These variants significantly decreased SL8-presenting SCT at the cell surface in the presence of ERAP1, but did not affect overall SCT expression, indicating that ERAP1 trims the SL8 N terminus. Comparison of the X-ray crystal structures of WT and three variant SCTs revealed only minor perturbations of the peptide-binding domain in the variants. However, molecular dynamics simulations suggested that SL8 can dissociate partially within a sub-microsecond timescale, exposing its N terminus to the solvent. We also found that the C terminus of MHC I–bound SL8 remains deeply buried in the F-pocket of MHC I. Furthermore, free-energy calculations revealed that the three SCT variants exhibit lower free-energy barriers of N terminus dissociation than the WT Kb. Taken together, our results are consistent with a previously observed model in which the partial dissociation of bound peptides from MHC I exposes their N terminus to trimming by ERAP1, whereas their C terminus is anchored at the F-pocket.

Background: Hepatocellular carcinoma (HCC) is a common and deadly cancer, however, very little improvement has been made towards its diagnosis and prognosis. The expression and functional contribution of the receptor tyrosine kinase ROR1 have not been investigated in HCC before. Hence, we investigated the expression of ROR1 in HCC cells and assessed its involvement in hepatocarcinogenesis. Methods: Recombinant bacterial ROR1 protein was used as an immunogen to generate ROR1 monoclonal antibodies. ROR1 transcript levels were detected by RT-qPCR and the protein expression of ROR1 in HCC was assessed by Western blotting by using homemade anti-ROR1 monoclonal antibodies. Apoptosis, cell cycle, trans-well migration, and drug efflux assays were performed in shRNA-ROR1 HCC cell clones to uncover the functional contribution of ROR1 to hepatocarcinogenesis. Results: New ROR1 antibodies specifically detected endogenous ROR1 protein in human and mouse HCC cell lines. ROR1-knockdown resulted in decreased proliferation and migration but enhanced resistance to apoptosis and anoikis. The observed chemotherapy-resistant phenotype of ROR1-knockdown cells was due to enhanced drug efflux and increased expression of multi-drug resistance genes. Conclusions: ROR1 is expressed in HCC and contributes to disease development by interfering with multiple pathways. Acquired ROR1 expression may have diagnostic and prognostic value in HCC.

Summary Anti-CD40 monoclonal antibodies (mAbs) comprise agonists and antagonists, which display promising therapeutic activities in cancer and autoimmunity, respectively. We previously showed that epitope and isotype interact to deliver optimal agonistic anti-CD40 mAbs. The impact of Fc engineering on antagonists, however, remains largely unexplored. Here, we show that clinically relevant antagonists used for treating autoimmune conditions can be converted into potent FcγR-independent agonists with remarkable antitumor activity by isotype switching to hIgG2. One antagonist is converted to a super-agonist with greater potency than previously reported highly agonistic anti-CD40 mAbs. Such conversion is dependent on the unique disulfide bonding properties of the hIgG2 hinge. This investigation highlights the transformative capacity of the hIgG2 isotype for converting antagonists to agonists to treat cancer., Graphical Abstract, Highlights • Antagonistic anti-CD40 mAbs can be converted into agonists by isotype switching to hIgG2 • Transformation is based upon the hIgG2 hinge • Transforms an antagonist to an agonist four times more potent than existing anti-CD40 mAbs • This converted antagonist exhibits antitumor synergy with cell therapy and vaccination, Yu et al. show that isotype switching can convert clinically relevant anti-CD40 antagonistic antibodies to potent FcγR-independent agonists. The converted antibodies can elicit strong antitumor responses in mouse models.

Background Castrate resistant prostate cancer (CRPC) is often driven by constitutively active forms of the androgen receptor such as the V7 splice variant (AR-V7) and commonly becomes resistant to established hormonal therapy strategies such as enzalutamide as a result. The lysine demethylase LSD1 is a co-activator of the wild type androgen receptor and a potential therapeutic target in hormone sensitive prostate cancer. We evaluated whether LSD1 could also be therapeutically targeted in CRPC models driven by AR-V7. Methods We utilised cell line models of castrate resistant prostate cancer through over expression of AR-V7 to test the impact of chemical LSD1 inhibition on AR activation. We validated findings through depletion of LSD1 expression and in prostate cancer cell lines that express AR-V7. Results Chemical inhibition of LSD1 resulted in reduced activation of the androgen receptor through both the wild type and its AR-V7 splice variant forms. This was confirmed and validated in luciferase reporter assays, in LNCaP and 22Rv1 prostate cancer cell lines and in LSD1 depletion experiments. Conclusion LSD1 contributes to activation of both the wild type and V7 splice variant forms of the androgen receptor and can be therapeutically targeted in models of CRPC. Further development of this approach is warranted. Electronic supplementary material The online version of this article (10.1186/s12935-018-0568-1) contains supplementary material, which is available to authorized users.

Vaccination with DNA that encodes cancer antigens is a simple and convenient way to raise immunity against cancer and has already shown promise in the clinical setting. Conventional plasmid DNA is commonly used which together with the encoded antigen also includes bacterial immunostimulatory CpG motifs to target the DNA sensor Toll-like receptor 9. Recently DNA vaccines using doggybone DNA (dbDNA™), have been developed without the use of bacteria. The cell-free process relies on the use of Phi29 DNA polymerase to amplify the template followed by protelomerase TelN to complete individual closed linear DNA. The resulting DNA contains the required antigenic sequence, a promoter and a poly A tail but lacks bacterial sequences such as an antibiotic resistance gene, prompting the question of immunogenicity. Here we compared the ability of doggybone DNA vaccine with plasmid DNA vaccine to induce adaptive immunity using clinically relevant oncotargets E6 and E7 from HPV. We demonstrate that despite the inability to trigger TLR9, doggybone DNA was able to induce similar levels of cellular and humoral immunity as plasmid DNA, with suppression of established TC-1 tumours. Electronic supplementary material The online version of this article (10.1007/s00262-017-2111-y) contains supplementary material, which is available to authorized users.

Attempts to generate robust anti-tumour cytotoxic T lymphocyte (CTL) responses using immunotherapy are frequently thwarted by exhaustion and anergy of CTL recruited to tumour. One strategy to overcome this is to retarget a population of virus-specific CTL to kill tumour cells. Here, we describe a proof-of-principle study using a bispecific conjugate designed to retarget ovalbumin (OVA)-specific CTL to kill tumour cells via CD20. A single-chain trimer (SCT) consisting of MHCI H-2K(b)/SIINFEKL peptide/beta 2 microglobulin/BirA was expressed in bacteria, refolded and chemically conjugated to one (1:1; F2) or two (2:1; F3) anti-hCD20 Fab' fragments. In vitro, the [SCT × Fab'] (F2 and F3) redirected SIINFEKL-specific OT-I CTL to kill CD20(+) target cells, and in the presence of CD20(+) target cells to provide crosslinking, they were also able to induce proliferation of OT-I cells. In vivo, activated OT-I CTL could be retargeted to kill [SCT × Fab']-coated B cells from hCD20 transgenic (hCD20 Tg) mice and also EL4 and B16 mouse tumour cells expressing human CD20 (hCD20). Importantly, in a hCD20 Tg mouse model, [SCT × Fab'] administered systemically were able to retarget activated OT-I cells to deplete normal B cells, and their performance matched that of a bispecific antibody (BsAb) comprising anti-CD3 and anti-CD20. [SCT × Fab'] were also active therapeutically in an EL4 tumour model. Furthermore, measurement of serum cytokine levels suggests that [SCT × Fab'] are associated with a lower level of inflammatory cytokine release than the BsAb and so may be advantageous clinically in terms of reduced toxicity.

We report a series of tranylcypromine analogues containing a fluorine in the cyclopropyl ring. A number of compounds with additional m- or p-substitution of the aryl ring were micromolar inhibitors of the LSD1 enzyme. In cellular assays, the compounds inhibited the proliferation of acute myeloid leukemia cell lines. Increased levels of the biomarkers H3K4me2 and CD86 were consistent with LSD1 target engagement.

The auxiliary α2δ subunits of voltage-gated calcium channels are extracellular membrane-associated proteins, which are post-translationally cleaved into disulfide-linked polypeptides α2 and δ. We now show, using α2δ constructs containing artificial cleavage sites, that this processing is an essential step permitting voltage-dependent activation of plasma membrane N-type (CaV2.2) calcium channels. Indeed, uncleaved α2δ inhibits native calcium currents in mammalian neurons. By inducing acute cell-surface proteolytic cleavage of α2δ, voltage-dependent activation of channels is promoted, independent from the trafficking role of α2δ. Uncleaved α2δ does not support trafficking of CaV2.2 channel complexes into neuronal processes, and inhibits Ca2+ entry into synaptic boutons, and we can reverse this by controlled intracellular proteolytic cleavage. We propose a model whereby uncleaved α2δ subunits maintain immature calcium channels in an inhibited state. Proteolytic processing of α2δ then permits voltage-dependent activation of the channels, acting as a checkpoint allowing trafficking only of mature calcium channel complexes into neuronal processes. DOI: http://dx.doi.org/10.7554/eLife.21143.001

Voltage-gated calcium channels are thought to exist in the plasma membrane as heteromeric proteins, in which the α1 subunit is associated with two auxiliary subunits, the intracellular β subunit and the α 2 δ subunit; both of these subunits influence the trafficking and properties of Ca V 1 and Ca V 2 channels. The α 2 δ subunits have been described as type I transmembrane proteins, because they have an N-terminal signal peptide and a C-terminal hydrophobic and potentially transmembrane region. However, because they have very short C-terminal cytoplasmic domains, we hypothesized that the α 2 δ proteins might be associated with the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor attached to δ rather than a transmembrane domain. Here, we provide biochemical, immunocytochemical, and mutational evidence to show that all of the α 2 δ subunits studied, α 2 δ-1, α 2 δ-2, and α 2 δ-3, show all of the properties expected of GPI-anchored proteins, both when heterologously expressed and in native tissues. They are substrates for prokaryotic phosphatidylinositol-phospholipase C (PI-PLC) and trypanosomal GPI-PLC, which release the α 2 δ proteins from membranes and intact cells and expose a cross-reacting determinant epitope. PI-PLC does not affect control transmembrane or membrane-associated proteins. Furthermore, mutation of the predicted GPI-anchor sites markedly reduced plasma membrane and detergent-resistant membrane localization of α 2 δ subunits. We also show that GPI anchoring of α 2 δ subunits is necessary for their function to enhance calcium currents, and PI-PLC treatment only reduces calcium current density when α 2 δ subunits are coexpressed. In conclusion, this study redefines our understanding of α 2 δ subunits, both in terms of their role in calcium-channel function and other roles in synaptogenesis.

Neuropathic pain results from damage to the peripheral sensory nervous system, which may have a number of causes. The calcium channel subunit α2δ-1 is upregulated in dorsal root ganglion (DRG) neurons in several animal models of neuropathic pain, and this is causally related to the onset of allodynia, in which a non-noxious stimulus becomes painful. The therapeutic drugs gabapentin and pregabalin (PGB), which are both α2δ ligands, have antiallodynic effects, but their mechanism of action has remained elusive. To investigate this, we used anin vivorat model of neuropathy, unilateral lumbar spinal nerve ligation (SNL), to characterize the distribution of α2δ-1 in DRG neurons, both at the light- and electron-microscopic level. We found that, on the side of the ligation, α2δ-1 was increased in the endoplasmic reticulum of DRG somata, in intracellular vesicular structures within their axons, and in the plasma membrane of their presynaptic terminals in superficial layers of the dorsal horn. Chronic PGB treatment of SNL animals, at a dose that alleviated allodynia, markedly reduced the elevation of α2δ-1 in the spinal cord and ascending axon tracts. In contrast, it had no effect on the upregulation of α2δ-1 mRNA and protein in DRGs.In vitro, PGB reduced plasma membrane expression of α2δ-1 without affecting endocytosis. We conclude that the antiallodynic effect of PGBin vivois associated with impaired anterograde trafficking of α2δ-1, resulting in its decrease in presynaptic terminals, which would reduce neurotransmitter release and spinal sensitization, an important factor in the maintenance of neuropathic pain.

CaV1 and CaV2 voltage-gated calcium channels contain an α1 subunit, which forms the selective pore and determines the functional properties of the channel. The α1 subunit is associated with auxiliary β and α2δ subunits, which modulate the trafficking and functional properties of the channels.α2δ subunits are membrane-associated proteins, which are highly glycosylated and possess multiple disulfide bonds. The α2 and δ peptides are encoded by a single gene as an uninterrupted α2δ pre-protein, which is cleaved post-translationally by a yet unidentified protease. The highly glycosylated α2 polypeptide, which is entirely extracellular, remains disulfide-bonded to the smaller δ moiety, which links the protein to the plasma membrane. All four known α2δ subtypes are cleaved into α2 and δ in vivo, but the significance of the proteolytic processing with regard of the function of α2δs as voltage-gated calcium channel subunits has remained elusive.We now provide evidence that preventing the cleavage, by mutating the proteolytic site in α2δ-1 and α2δ-3 subtypes, abolishes any effect of α2δ on CaV currents in tsA −201 cells expressing CaV2.2/β. In contrast, our experiments indicate that the trafficking of Cav2.2 channels by α2δ-1 is not affected by the lack of cleavage. In neurons, we find that all α2δ-1 in DRG neuronal axons is proteolytically cleaved; however, in cell bodies not all α2δ-1 is cleaved, indicating that there is a potential modulatory role for unprocessed α2δ.We are currently examining the nature of the protease(s) involved in the processing of α2δ-1 by application of selected chemical protease inhibitors.Targeting the protease responsible for the cleavage of α2δ-1 could provide a novel avenue for therapy of neuropathic pain, which would act on the same target as gabapentinoids, but by a completely different mechanism.

The accessory alpha2delta subunits of voltage-gated calcium channels are type 1 transmembrane proteins that are highly glycosylated and possess multiple disulfide bonds. From studies of the topology and processing of skeletal-muscle alpha2delta-1, it has been shown to be post-translationally cleaved into an alpha2 and a delta subunit, which remain disulfide-bonded. In the present study, we have examined the processing of alpha2delta-2 subunits when stably or transiently expressed, in tsA (temperature-sensitive A)-201, Cos-7 and NG108-15 cells, and compared it with that observed in the cerebellum. Despite showing full functionality and being expressed on the plasma membrane, the vast majority of heterologously expressed alpha2delta-2 is not cleaved into alpha(2)-2 and delta-2, unlike endogenous alpha2delta-2 in the cerebellum. It remains an open question for future research whether alpha2delta-2 is functional in its calcium channel trafficking role in its proteolytically cleaved or non-cleaved state.

All auxiliary α 2 δ subunits of voltage-gated Ca 2+ (Ca V ) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in α 2 δ-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the α 2 δ-2 VWA domain shows it to be highly likely to bind a divalent cation. Mutating the three key MIDAS residues responsible for divalent cation binding resulted in a MIDAS mutant α 2 δ-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT α 2 δ-2, the MIDAS mutant α 2 δ-2 subunit did not enhance and, in some cases, further diminished Ca V 1.2, -2.1, and -2.2 currents coexpressed with β1b by using either Ba 2+ or Na + as a permeant ion. Furthermore, expression of the MIDAS mutant α 2 δ-2 reduced surface expression and strongly increased the perinuclear retention of Ca V α1 subunits at the earliest time at which expression was observed in both Cos-7 and NG108–15 cells. Despite the presence of endogenous α 2 δ subunits, heterologous expression of α 2 δ-2 in differentiated NG108–15 cells further enhanced the endogenous high-threshold Ca 2+ currents, whereas this enhancement was prevented by the MIDAS mutations. Our results indicate that α 2 δ subunits normally interact with the Ca V α1 subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the α 2 δ subunit is required to promote trafficking of the α1 subunit to the plasma membrane by an integrin-like switch. This finding provides evidence for a primary role of a VWA domain in intracellular trafficking of a multimeric complex, in contrast to the more usual roles in binding extracellular ligands in other exofacial VWA domains.

STK15/Aurora2 is a centrosome-associated serine/threonine kinase, the protein levels and kinase activity of which rise during G2 and mitosis. STK15 overexpression induces tumorigenesis and is amplified in various human cancers and tumor cell lines. Thus, STK15 represents an important therapeutic target for small molecule inhibitors that would disrupt its activity and block cell proliferation. The availability of a robust and selective small molecule inhibitor would also provide a useful tool for identification of the potential role of STK15 in cell cycle regulation and tumor development. The authors report the development of a novel, fast, simple microplate assay for STK15 activity suitable for high-throughput screening. In the assay, gamma-(33)P-ATP and STK15 were incubated in a myelin basic protein (MBP)-coated FlashPlate(R) to generate a scintillation signal. The assay was reproducible, the signal-to-noise ratio was high (11) and the Z' factor was 0.69. The assay was easily adapted to a robotic system for drug discovery programs targeting STK15. The authors also demonstrate that STK15 is regulated by phosphorylation and the N-amino terminal domain of the protein. Treatment with phosphatase inhibitors (okadaic acid) or deletion of the N-amino terminal domain results in a significant increase in the enzymatic activity.

The High Voltage-Activated (HVA) subgroup of voltage-gated calcium channels contain an α1 subunit, which forms the selective pore and determines the main functional properties of the channel. The α1 subunit is associated with auxiliary subunits including β and α2δ, which modulate trafficking and functional properties of the channels.There are four known genes encoding α2δ subunits, which are believed to have similar structure. They consist of two peptides: the highly glycosylated α2 which is entirely extracellular is disulfide-bonded to a δ subunit that links the protein into the plasma membrane. The α2 and δ peptides are encoded by a single gene as an uninterrupted α2δ pre-protein, which is further processed post-translationally. We have recently shown that α2δ subunits are glycosyl phophatidyl inositol (GPI)-achored proteins (Davies et al., 2010, PNAS 107:1654-1659), and this is essential for their function, and explains their localization in lipid raft fractions (Davies et al, 2006, J. Neurosci. 26: 8748-8757).We further studied the mechnism and consequences of the proteolytic cleavage of the α2δ subunits (α2δ-1, -2 and -3)and identified the cleavage sites in α2δ-2 and α2δ-3. Western blots from brain tissue revealed only the mature form of the protein strongly associated with lipid rafts. However, transfecting mammalian cells with cDNA for α2δ-1,-2,and -3 resulted in incomplete cleavage (∼ 40-60% processing) most probably due to limitations of the cleavage-mediating protease(s) in heterologous expression systems. The mature form is localized in lipid rafts, suggesting that maturation of the protein might occur in localized membrane domains. Moreover, electrophysiological recordings demonstrated that proteolytic cleavage is key to the function of these subunits.We are currently examining the nature of the protease(s) involved in this proteolytic processing.

In this review, we examine what is known about the mechanism of action of the auxiliary alpha2delta subunits of voltage-gated Ca(2+) (Ca(v)) channels. First, to provide some background on the alpha2delta proteins, we discuss the genes encoding these channels, in addition to the topology and predicted structure of the alpha2delta subunits. We then describe the effects of alpha2delta subunits on the biophysical properties of Ca(v) channels and their physiological function. All alpha2delta subunits increase the density at the plasma membrane of Ca(2+) channels activated by high voltage, and we discuss what is known about the mechanism underlying this trafficking. Finally, we consider the link between alpha2delta subunits and disease, both in terms of spontaneous and engineered mouse mutants that show cerebellar ataxia and spike-wave epilepsy, and in terms of neuropathic pain and the mechanism of action of the gabapentinoid drugs - small-molecule ligands of the alpha2delta-1 and alpha2delta-2 subunits.

The accessory α2δ subunits of voltage-gated calcium channels are highly glycosylated transmembrane proteins that interact with calcium channel α1 subunits to enhance calcium currents. We compared the membrane localization and processing of native cerebellar α2δ-2 subunits with α2δ-2 stably expressed in tsA-201 cells. We identified that α2δ-2 is completely concentrated in cholesterol-rich microdomains (lipid rafts) in cerebellum, in which it substantially colocalizes with the calcium channel α1 subunit CaV2.1, although CaV2.1 is also present in the Triton X-100-soluble fraction. In tsA-201 cells, unlike cerebellum, α2δ-2 is not completely proteolytically processed into α2-2 and δ-2. However, this processing is more complete in the lipid raft fraction of tsA-201 cells, in which α2δ-2 also colocalizes with CaV2.1. Cholesterol depletion of intact cells disrupted their lipid rafts and enhanced CaV2.1/α2δ-2/β4 currents. Furthermore, α2δ-2 coimmunoprecipitates with lipid raft-associated proteins of the stomatin family. The apparent affinity of α2δ-2 for its ligand gabapentin is increased markedly in the cholesterol-rich microdomain fractions, in both cerebellum and the stable α2δ-2 cell line. In contrast, α2δ-2 containing a point mutation (R282A) has a much lower affinity for gabapentin, and this is not enhanced in the lipid raft fraction. This R282A mutant α2δ-2 shows reduced functionality in terms of enhancement of CaV2.1/β4 calcium currents, suggesting that the integrity of the gabapentin binding site may be important for normal functioning of α2δ-2. Together, these results indicate that both α2δ-2 and CaV2.1 are normally associated with cholesterol-rich microdomains, and this influences their functionality.

The High Voltage-Activated (HVA) subgroup of voltage-gated calcium channels contain an α1 subunit, which forms the selective pore and determines the main functional properties of the channel. The α1 subunit is associated with auxiliary subunits including intracellular β and α2δ, which modulate trafficking and functional properties of the channels.α2δ subunits consists of two peptides: α2 which is entirely extracellular is disulfide-bonded to a δ subunit that links the protein into the plasma membrane. There are four genes encoding α2δ subunits, which are believed to have similar structure. We have shown previously that α2δ-2 subunits associate with lipid rafts, that are sub-domains of the cell membrane enriched in cholesterol and glycosphingolipids.We have addressed the ability of α2δ-3 to associate with lipid rafts in both native tissues (it is highly expressed in brain) and in overexpression systems. We have generated mutations which reduced expression of the subunit in lipid rafts as well as the surface expression of the protein. These mutations reduced the enhancing effect of α2δ-3 on calcium channel currents.The α2 and δ peptides are product of a single gene, and they are encoded as an uninterrupted α2δ pre-protein, which is further processed post-translationally. In native tissues we observed exclusively the mature form of the protein, which was strongly associated with the lipid rafts. However, in several overexpression systems we could also detect unprocessed α2δ-3 pre-protein coexisting with the mature α2δ3. The unprocessed form was localized both in the rafts and non-raft protein fractions, suggesting that maturation of the protein might occur in localized membrane domains.These results further demonstrate the role of lipid rafts in the regulation of Ca channel currents by α2δ and their involvement in the maturation of the α2δ protein.