Functional characterisation of the R2452W ryanodine receptor variant associated with malignant hyperthermia susceptibility

Malignant hyperthermia (MH) is a pharmacogenetic disorder that manifests in susceptible individuals exposed to volatile anaesthetics. Over 400 variants in the ryanodine receptor 1 (RYR1) have been reported but relatively few have been definitively associated with susceptibility to MH. This is largely due to the technical challenges of demonstrating abnormal Ca²⁺ release from the sarcoplasmic reticulum. This study focuses on the R2452W variant and its functional characterisation with the aim of classifying this variant as MH causative. HEK293 cells were transiently transfected with full-length human wildtype or R2452W mutant RYR1 cDNA. In addition, B-lymphoblastoid cells from blood and myoblasts propagated from in vitro contracture tests were extracted from patients positive for the R2452W variant. All cell lines generated were loaded with the ratiometric dye Fura-2 AM, stimulated with the RYR1-specific agonist 4-chloro-m-cresol and Ca²⁺ release from the sarcoplasmic/endoplasmic reticulum was monitored by fluorescence emission. All cells expressing the RYR1 R2452W variant show a significantly higher Ca²⁺ release in response to the agonist, 4-chloro-m-cresol, compared to cells expressing RYR1 WT. These results indicate that the R2452W variant results in a hypersensitive ryanodine receptor 1 and suggest that the R2452W variant in the ryanodine receptor 1 is likely to be causative of MH.     

Development of a functional assay for Ca2+ release activity of IP3R and expression of an IP3R gene fragment in the baculovirus-insect cell system

Receptor-stimulated phosphoinositide (PI) hydrolysis is an important and ubiquitous mechanism of intracellular signaling. Inositol 1,4,5-trisphosphate (IP₃), generated by phosphoinositide (PI) hydrolysis, binds to and gates an intracellular Ca²⁺ channel, the IP₃ receptor (IP₃R), which is therefore a central component of this signaling cascade. Here we describe the development of a baculovirus (BV)/Sf(S. frugiperda) cell system that can be used to look at IP₃R function. Agonist-evoked changes in intracellular Ca²⁺ levels [Ca²⁺]_i were measured (using Fura2) in Sf cells expressing the gene encoding the muscarinic acetylcholine receptor (vmlAchR). Furthermore, we have constructed a recombinant BV (vlP₃R), with the core of the IP₃R ligand-binding domain from the Drosophila IP₃R, under the polyhedrin promoter. The recombinant protein from such a virus was expected to act as a large ligand sink for IP3, generated by stimulation of vmlAchR. Cells coinfected with recombinant BV carrying the potential dominant-negative vIP₃R construct and vmlAchR have been used to assay the modulation of IP₃R-mediated Ca²⁺ release, by the ligand sink.     

Effects of IP<Subscript>3</Subscript>R2 Receptor Deletion in the Ischemic Mouse Retina

Glial cells in the diseased nervous system undergo a process known as reactive gliosis. Gliosis of retinal Müller glial cells is characterized by an upregulation of glial fibrillary acidic protein and frequently by a reduction of inward K⁺ current amplitudes. Purinergic signaling is assumed to be involved in gliotic processes. As previously shown, lack of the nucleotide receptor P2Y₁ leads to an altered regulation of K⁺ currents in Müller cells of the ischemic retina. Here, we asked first whether this effect is mediated by the IP₃ receptor subtype 2 (IP₃R2) known as the major downstream signaling target of P2Y₁ in Müller cells. The second question was whether lack of IP₃R2 affects neuronal survival in the control and ischemic retina. Ischemia was induced in wild type and IP₃R2-deficient (IP ₃ R2 ^?/?) mice by transient elevation of the intraocular pressure. Immunostaining and TUNEL labelling were used to quantify neuronal cell loss. The downregulation of inward K⁺ currents in Müller cells from ischemic IP ₃ R2 ^?/? retinae was less strong than in wild type animals. The reduction of the number of cells in the ganglion cell layer and of calretinin- and calbindin-positive cells 7 days after ischemia was similar in wild type and IP ₃ R2 ^?/? mice. However, IP₃R2 deficiency led to an increased number of TUNEL-positive cells in the outer nuclear layer at 1 day and to an enhanced postischemic loss of photoreceptors 7 days after ischemia. This implies that IP₃R2 is involved in some but not all aspects of signaling in Müller cells after an ischemic insult.     

Caspase-dependent Proteolysis of Human Ribonucleotide Reductase Small Subunits R2 and p53R2 during Apoptosis

Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has also been suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp³⁴² and Asn³⁴³. Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspase-3 and -8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7, and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, which was prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis.     

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 10⁵/μg of DNA to 10⁷/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination.     

CD200R1 Supports HSV-1 Viral Replication and Licenses Pro-Inflammatory Signaling Functions of TLR2

The CD200R1:CD200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. We generated CD200R1^−/− mice and employed them to explore both the role of CD200R1 in regulating macrophage signaling via TLR2 as well as the host response to an in vivo, TLR2-dependent model, herpes simplex virus 1 (HSV-1) infection. CD200R1^−/− peritoneal macrophages demonstrated a 70–75% decrease in the generation of IL-6 and CCL5 (Rantes) in response to the TLR2 agonist Pam₂CSK₄ and to HSV-1. CD200R1^−/− macrophages could neither up-regulate the expression of TLR2, nor assemble a functional inflammasome in response to HSV-1. CD200R1^−/− mice were protected from HSV-1 infection and exhibited dysfunctional TLR2 signaling. Finally, both CD200R1^−/− mice and CD200R1^−/− fibroblasts and macrophages showed a markedly reduced ability to support HSV-1 replication. In summary, our data demonstrate an unanticipated and novel requirement for CD200R1 in “licensing” pro-inflammatory functions of TLR2 and in limiting viral replication that are supported by ex vivo and in vivo evidence.     

Epitope-Specific Mechanisms of IGF1R Inhibition by Ganitumab

Background

Therapeutic antibodies targeting the IGF1R have shown diverse efficacy and safety signals in oncology clinical trials. The success of these agents as future human therapeutics depends on understanding the specific mechanisms by which these antibodies target IGF1R signaling.

Methodology/Principal Findings

A panel of well-characterized assays was used to investigate the mechanisms by which ganitumab, a fully human anti-IGF1R antibody undergoing clinical testing, inhibits IGF1R activity. Epitope mapping using IGF1R subdomains localized the ganitumab binding site to the L2 domain. Binding of ganitumab inhibited the high-affinity interaction of IGF-1 and IGF-2 required to activate IGF1R in cells engineered for IGF1R hypersensitivity and in human cancer cell lines, resulting in complete blockade of ligand-induced cellular proliferation. Inhibition of IGF1R activity by ganitumab did not depend on endosomal sequestration, since efficient ligand blockade was obtained without evidence of receptor internalization and degradation. Clinically relevant concentrations of ganitumab also inhibited the activation of hybrid receptors by IGF-1 and IGF-2. Ganitumab was not an agonist of homodimeric IGF1R or hybrid receptors in MCF-7 and COLO 205 cells, but low-level IGF1R activation was detected in cells engineered for IGF1R hypersensitivity. This activation seems biologically irrelevant since ganitumab completely inhibited ligand-driven proliferation. The in vivo efficacy profile of ganitumab was equivalent or better than CR and FnIII-1 domain-specific antibodies, alone or in combination with irinotecan. CR domain-specific antibodies only blocked IGF-1 binding to IGF1R but were more potent than ganitumab at inducing homodimer and hybrid receptor downregulation in vitro, however this difference was less obvious in vivo. No inhibition of hybrid receptors was observed with the FnIII-1 domain antibodies, which were relatively strong homodimer and hybrid agonists.

Conclusions/Significance

The safety and efficacy profile of ganitumab and other anti-IGF1R antibodies may be explained by the distinct molecular mechanisms by which they inhibit receptor signaling.     

Aberrant CD200/CD200R1 expression and function in systemic lupus erythematosus contributes to abnormal T-cell responsiveness and dendritic cell activity

Introduction

CD200 is a type I transmembrane glycoprotein that can regulate the activation threshold of inflammatory immune responses, polarize cytokine production, and maintain immune homeostasis. We therefore evaluated the functional status of CD200/CD200 receptor 1 (CD200R1) interactions in subjects with systemic lupus erythematosus (SLE).

Methods

Serum CD200 level was detected by ELISA. The expression of CD200/CD200R1 by CD4⁺ T cells and dendritic cells (DCs) was examined by flow cytometry, and then compared between SLE patients and healthy controls. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidyl ester and annexin V/propidium iodide for evaluation of the effect of CD200 on cell proliferation and apoptosis. In addition, the effect of CD200 on DC function was determined by transwell migration assay as well as by measurement of binding and phagocytosis of apoptotic cells.

Results

In SLE patients, the number of CD200⁺ cells and the level of soluble CD200 were significantly higher than in healthy controls, whereas the expression of CD200R1 by CD4⁺ T cells and DCs was decreased. Furthermore, the increased CD200 expression by early apoptotic cells contributed to their diminished binding and phagocytosis by DCs in SLE. Importantly, the engagement of CD200 receptor on CD4⁺ T cells with CD200-Fc fusion protein in vitro reduced the differentiation of T-helper type 17 cells and reversed the defective induction of CD4⁺CD25^highFoxP3⁺ T cells by transforming growth factor beta in SLE patients. Conversely, blockade of CD200-CD200R1 interaction with anti-CD200R1 antibody promoted CD4⁺ T-cell proliferation.

Conclusion

CD200 and CD200R1 expression and function are abnormal in SLE and may contribute to the immunologic abnormalities in SLE.     

Multiple Autoimmune-Associated Variants Confer Decreased IL-2R Signaling in CD4+CD25hi T Cells of Type 1 Diabetic and Multiple Sclerosis Patients

IL-2 receptor (IL-2R) signaling is essential for optimal stability and function of CD4⁺CD25^hiFOXP3⁺ regulatory T cells (Treg); a cell type that plays an integral role in maintaining tolerance. Thus, we hypothesized that decreased response to IL-2 may be a common phenotype of subjects who have autoimmune diseases associated with variants in the IL2RA locus, including T1D and MS, particularly in cells expressing the high affinity IL-2R alpha chain (IL-2RA or CD25). To examine this question we used phosphorylation of STAT5 (pSTAT5) as a downstream measure of IL-2R signaling, and found a decreased response to IL-2 in CD4⁺CD25^hi T cells of T1D and MS, but not SLE patients. Since the IL2RArs2104286 haplotype is associated with T1D and MS, we measured pSTAT5 in controls carrying the rs2104286 risk haplotype to test whether this variant contributed to reduced IL-2 responsiveness. Consistent with this, we found decreased pSTAT5 in subjects carrying the rs2104286 risk haplotype. Reduced IL-2R signaling did not result from lower CD25 expression on CD25^hi cells; instead we detected increased CD25 expression on naive Treg from controls carrying the rs2104286 risk haplotype, and subjects with T1D and MS. However the rs2104286 risk haplotype correlated with increased soluble IL-2RA levels, suggesting that shedding of the IL-2R may account in part for the reduced IL-2R signaling associated with the rs2104286 risk haplotype. In addition to risk variants in IL2RA, we found that the T1D-associated risk variant of PTPN2rs1893217 independently contributed to diminished IL-2R signaling. However, even when holding genotype constant at IL2RA and PTPN2, we still observed a significant signaling defect in T1D and MS patients. Together, these data suggest that multiple mechanisms converge in disease leading to decreased response to IL-2, a phenotype that may eventually lead to loss of tolerance and autoimmunity.     

Complementation analysis in Pseudomonas aeruginosa of the transfer genes of the wide host range R plasmid R18

A method of transductional complementation was developed in Pseudomonas aeruginosa to identify the cistrons involved in the conjugal transfer of the wide host range R plasmid R18. This used the P. aeruginosa bacteriophage E79tv-2 and has led to the identification of eight tra cistrons encoded by this plasmid. Plasmids mutant in six cistrons, traA, traB, traC, traD, traE, and traG were resistant to donor-specific phage (Dps^?) while traF and traH mutant plasmids retained phage sensitivity. Some traB mutants were unable to inhibit the replication of phage G101 (Phi(G101)^?) while some were also deficient in entry exclusion (Eex^?). Two traB mutants which were also Eex^? were suppressible by an amber suppressor. Three tra mutants selected directly as being Phi(G101)^? were found to be also Dps^?Eex^? and mutant in traB. These data suggest a relationship between traB, Eex, and Phi(G101). In order to facilitate future genetic comparison of the tra genes of R18 and other wide host range plasmids and the role of the host in conjugation, R18 DNA was compared with that of RP4, by restriction enzyme fragment patterns and found to be identical.     

A knock-in mouse model of N-terminal R420W mutation of cardiac ryanodine receptor exhibits arrhythmogenesis with abnormal calcium dynamics in cardiomyocytes

Cardiac ryanodine receptor gene (RyR2) mutations cause fatal arrhythmogenic diseases such as catecholaminergic polymorphic ventricular tachycardia and arrhythmogenic right ventricular cardiomyopathy. The N-terminal region of RyR2 is one of the hot spots for mutations. In this study, we investigated cardiac phenotypes of a knock-in mouse model carrying R420W mutation of RyR2. The N-terminal R420W mutation has already been found in juvenile sudden death cadavers of unrelated families. The depolarization-induced Ca²⁺ transient amplitude was significantly lower in cardiomyocytes from RyR2^R420W/R420W mice compared with wild-type mice. The time to peak of the Ca²⁺ transient was significantly increased in RyR2^R420W/R420W mice. Furthermore, the prolonged decay time from the peak of the Ca²⁺ transient was detected in RyR2^R420W/R420W mice. ECG telemetry revealed that various types of arrhythmias were induced in RyR2^R420W/R420W mice in response to administration of caffeine and adrenaline. The mutant mice showed high occurrences of arrhythmias in response to heart stimulants compared with wild-type mice. These findings suggest that R420W mutation impairs depolarization-induced Ca²⁺ oscillation in cardiomyocytes, which possibly results in sudden death due to stress-induced arrhythmias.     

Coordination of Sex Pili with their Specifying R Factors

A single bacterial cell can simultaneously carry both F-like (fi⁺) and I-like (fi^?) R factors and, when the R factors are de-repressed, most cells produce both F-like and I-like sex pili. These pili can be distinguished immunologically and by their capacity to adsorb different phages¹. The F pilus is the receptor for RNA phages such as MS2 and filamentous DNA phages such as M13. The I pilus is the receptor for other filamentous DNA phages such as If1 and If2. Electron microscopy suggests that these filamentous DNA phages, both F-specific and I-specific, adsorb to the tip of the pilus^2,3.     

An IP3R3- and NPY-Expressing Microvillous Cell Mediates Tissue Homeostasis and Regeneration in the Mouse Olfactory Epithelium

Calcium-dependent release of neurotrophic factors plays an important role in the maintenance of neurons, yet the release mechanisms are understudied. The inositol triphosphate (IP3) receptor is a calcium release channel that has a physiological role in cell growth, development, sensory perception, neuronal signaling and secretion. In the olfactory system, the IP3 receptor subtype 3 (IP3R3) is expressed exclusively in a microvillous cell subtype that is the predominant cell expressing neurotrophic factor neuropeptide Y (NPY). We hypothesized that IP3R3-expressing microvillous cells secrete sufficient NPY needed for both the continual maintenance of the neuronal population and for neuroregeneration following injury. We addressed this question by assessing the release of NPY and the regenerative capabilities of wild type, IP3R3^+/−, and IP3R3^−/− mice. Injury, simulated using extracellular ATP, induced IP3 receptor-mediated NPY release in wild-type mice. ATP-evoked NPY release was impaired in IP3R3^−/− mice, suggesting that IP3R3 contributes to NPY release following injury. Under normal physiological conditions, both IP3R3^−/− mice and explants from these mice had fewer progenitor cells that proliferate and differentiate into immature neurons. Although the number of mature neurons and the in vivo rate of proliferation were not altered, the proliferative response to the olfactotoxicant satratoxin G and olfactory bulb ablation injury was compromised in the olfactory epithelium of IP3R3^−/− mice. The reductions in both NPY release and number of progenitor cells in IP3R3^−/− mice point to a role of the IP3R3 in tissue homeostasis and neuroregeneration. Collectively, these data suggest that IP3R3 expressing microvillous cells are actively responsive to injury and promote recovery.     

Antigen-Presenting Cells Represent Targets for R5 HIV-1 Infection in the First Trimester Pregnancy Uterine Mucosa

Background

During the first trimester of pregnancy, HIV-1 mother-to-child transmission is relatively rare despite the permissivity of placental cells to cell-to-cell HIV-1 infection. The placenta interacts directly with maternal uterine cells (decidual cells) but the physiological role of the decidua in the control of HIV-1 transmission and whether decidua could be a source of infected cells is unknown.

Methodology/Principal Findings

To answer to this question, decidual mononuclear cells were exposed to HIV-1 in vitro. Decidual cells were shown to be more susceptible to infection by an R5 HIV-1, as compared to an X4 HIV-1. Infected cells were identified by flow cytometry analysis. The results showed that CD14⁺ cells were the main targets of HIV-1 infection in the decidua. These infected CD14⁺ cells expressed DC-SIGN, CD11b, CD11c, the Fc gamma receptor CD16, CD32 and CD64, classical MHC class-I and class-II and maturation and activation molecules CD83, CD80 and CD86. The permissivity of decidual tissue was also evaluated by histoculture. Decidual tissue was not infected by X4 HIV-1 but was permissive to R5 HIV-1. Different profiles of infection were observed depending on tissue localization.

Conclusions/Significance

The presence of HIV-1 target cells in the decidua in vitro and the low rate of in utero mother-to-child transmission during the first trimester of pregnancy suggest that a natural control occurs in vivo limiting cell-to-cell infection of the placenta and consequently infection of the fetus.     

The presence of LHRH-like receptors in Dunning R3327H prostate tumors

Quantitative analyses of LH-RH-like membrane receptors were performed in five tumors from the transplantable Dunning R3372H rat prostatic adenocarcinoma. The binding of D-Trp⁶-LH-RH, an agonist of LH-RH, was observed in all 5 tumors. The antagonist [Ac-Dp-Cl-Phe^1,2,D-Trp³,D-Lys⁶,D-Ala¹⁰]-LH-RH was bound to 4 tumors. The apparent equilibrium dissociation constant (K_d) for D-Trp⁶-LH-RH receptor was from 2.6–3.9 × 10^?10 M. The apparent equilibrium B_max values (maximum number of binding sites) were from 17.2–86.0 fmol/mg membrane protein for D-Trp⁶-LH-RH receptor. The K_d for the antagonist was from 2.4–2.7 × 10^?10 M and the B_max values were from 35.5–66.0 fmol/mg membrane protein. Similar binding studies performed in 6 normal rat prostates showed no binding capacities.     

Analysis of IS21-mediated mobilization of plasmid pACYC184 by R68.45 in Escherichia coli

Escherichia coli plasmids like pACYC184 or pBR325 can be mobilized by the P-type plasmid R68.45, which carries a tandem duplication of insertion element IS21, at a frequency of 10^?3–10^?5 per donor cell. Analysis of exconjugant cells revealed that plasmid mobilization occurs via cointegrate formation involving transposition of IS21. No resolution of cointegrates of pACYC184 and the P-type plasmid could be found in recA recipient cells. In the cointegrate, the E. coli plasmid is flanked by single copies of IS21 in direct orientation. After resolution of the cointegrate in recA⁺ recipients, the mobilizing plasmid R68.45 lost one copy of IS21 becoming indistinguishable from plasmid R68. It was shown that during mobilization, insertion element IS21 transposes to the mobilized plasmid. Insertion sites and orientations of IS21 in 33 pACYC184::IS21 insertion mutants have been determined: IS21 was found to be integrated in plasmid pACYC184 in different regions but only in one orientation. The IS21 tandem structure of plasmid R68.45 and its role in the mobilization process is discussed.     

The application of solid-state NMR spectroscopy to study candesartan cilexetil (TCV-116) membrane interactions. Comparative study with the AT1R antagonist drug olmesartan

ΑΤ1 receptor (AT1R) antagonists exert their antihypertensive effects by preventing the vasoconstrictive hormone AngII to bind to the AT1 receptor. It has been proposed that these biological effects are mediated through a two-step mechanism reaction. In the first step, they are incorporated in the core of the lipid bilayers and in the second step they reach the active site of the receptor through lateral diffusion. In this model, drug/membrane interactions are key elements for the drugs achieving inhibition at the AT1 receptor. In this work, the interactions of the prodrug candesartan cilexetil (TCV-116) with lipid bilayers are studied at molecular detail. Solid-state ¹³C-CP/MAS, 2D ¹H-¹H NOESY NMR spectroscopy and in silico calculations are used. TCV-116 and olmesartan, another drug which acts as an AT1R antagonist are compared for their dynamic effects in lipid bilayers using solid-state ²H-NMR. We find a similar localization of TCV-116 compared to other AT1 antagonists in the intermediate polar region. In addition, we can identify specific local interactions. These interactions may be associated in part with the discrete pharmacological profiles observed for different antagonists.     

Autoantibodies in primary Sjögren's syndrome patients induce internalization of muscarinic type 3 receptors

Objectives. Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by lymphocyte infiltration into the salivary and lachrymal glands, leading to dry mouth and eyes. The presence of functional autoantibodies against muscarinic type 3 receptor (M3R) has been reported in pSS patients. However, the pathological role of anti-M3R autoantibodies in pSS salivary dysfunction remains controversial. Methods. Purified IgGs were obtained from normal (control) and primary SS patients' sera (pSS IgG). Internalization of M3R and clathrin was analyzed by biochemical assay and immunofluorescence confocal microscopy using human submandibular gland (hSMG) cells. Cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) was measured by microspectrofluorimetry. Results. Incubation of hSMG cells with pSS IgG (1 mg/ml) significantly decreased M3R expression levels at the membrane. Carbachol-induced [Ca²⁺]_i transients (CICTs) in these cells were also inhibited by pSS IgG. In contrast to pSS IgG, control IgG had no effect on both the M3R expression level and CICTs. We found that binding of pSS IgG to M3R induces phosphorylation of the receptor, and that the pSS IgG-induced M3R internalization is prevented by the lysosomal inhibitor, chloroquine. In addition, pSS IgG decreased membrane clathrin expression, which was inhibited by atropine. Our immunofluorescence study further confirmed that pSS IgG induces a co-localization of M3R with clathrin and subsequent internalization of M3R. Conclusion. pSS IgG induces internalization of M3R partly through a clathrin-mediated pathway. The results suggest M3R internalization as a potential mechanism to explain the exocrinopathy seen in pSS patients.