Murine T cell proliferation can be specifically augmented by macrophages fed with specific antigen: alpha-2-macroglobulin conjugate

Foreign antigens conjugated to alpha-2-Macroglobulin (alpha-2-M) were effectively taken up by murine macrophages via alpha-2-M receptors. Such effective internalization of alpha-2-M:antigen conjugate by macrophages resulted in a remarkable increase in its ability to activate murine immune T cells under the following conditions. After macrophages were incubated with alpha-2-M:antigen conjugate or unconjugated antigen, they were cultured with immune T cells and antigen-stimulated tritiated thymidine incorporation by T cells was measured. The stimulation of T cell proliferative response by macrophages fed with the conjugate was sixteen times higher than what was observed with macrophages pretreated in the same concentration of unconjugated antigen. These findings suggest a physiological function of alpha-2-M and give us a new technique of immunization.     

The fate of internalized alpha-2-macroglobulin: alpha-galactosidase conjugate in fibroblasts from Fabry's hemizygote

In our previous report we described the endocytotic incorporation of coffee bean alpha-galactosidase conjugated to human alpha-2-macroglobulin (alpha-2-M) into cultured fibroblasts derived from a patient with Fabry's disease (1). The fate of internalized alpha-galactosidase according to the method described in the above report is now studied. Measurement of the enzyme activity of subcellular fractions showed that it was concentrated in the lysosomal-mitochondrial fraction. The half-life of internalized alpha-galactosidase was determined to be 2 h.     

Loss of electron-dense lamellar material from Fabry's disease fibroblasts after enzyme replacement

Cultured fibroblasts from a patient with Fabry's disease were treated with alpha-galactosidase A. The cells internalized the enzyme via a receptor-mediated transport system, resulting in the uptake of enzyme to 50% of the activity of normal cells. Following uptake of the enzyme and incubation for 9 days, a loss of electron-dense lamellar material within membrane-bound residual bodies was detected by electron microscopy. Morphometric analysis of electron micrographs showed that the percentage volume of cytoplasm occupied by electron-dense lamellar material in Fabry's disease fibroblasts decreased to near normal after treatment with enzyme. These results indicate that the ultrastructural abnormalities of Fabry's disease cells can be corrected by enzyme replacement, at least in cultured fibroblasts.     

Involvement of alpha-2-macroglobulin receptor in clearance of interleukin 8-alpha-2-macroglobulin complexes by human alveolar macrophages

The purpose of this study was to determine if interleukin 8 (IL-8) in complex with alpha2-macroglobulin (alpha-2-M) can be taken up by human alveolar macrophages. First, we demonstrated that human alveolar macrophages have receptors for alpha-2-M but not IL-8. The binding of(125)I-labeled alpha-2-M to the cells was specific and saturable, whereas(125)I-labeled recombinant human IL-8 (rhIL-8) did not bind to macrophages. However,(125)I-rhIL-8-alpha-2-M complexes bound to macrophages, and unlabeled alpha-2-M competed for the binding. We then cultured the cells in the presence of(125)I-rhIL-8-alpha-2-M complexes,(125)I-rhIL-8 alone or buffer for 24 h. Macrophages were lysed, and the released radioactivity measured. IL-8 concentrations in supernatants and cells were also measured using an IL-8 ELISA. When the macrophages were incubated with(125)I-rhIL-8-alpha-2-M complexes there was a significant amount of IL-8 associated with the cells. However, this was not the case when the cells were incubated with(125)I- rhIL-8 alone suggesting that only these complexes were taken-up by human alveolar macrophages. Furthermore, the clearance of complexes was specifically inhibited by a monoclonal antibody against the 515-kDa subunit of the alpha-2-M receptor (alpha-2-MR) but not by an isotopic mouse IgG1. The study shows an important clearance mechanism for IL-8 in the lung.     

Role of cleavage and shedding in human thyrotropin receptor function and trafficking.

The thyrotropin receptor (TSHR) undergoes a cleavage at the cell membrane, leading to a heterodimer, comprising an alpha extracellular and a beta-transmembrane and intracellular subunits, held together by disulfide bonds. Moreover, part of the alpha-subunit of the receptor is shed from thyroid and transfected L cells. To understand the role of cleavage and shedding, we constructed deletion mutants starting, respectively, at the most N-terminal (S314), and C-terminal (L378) cleavage sites previously mapped, corresponding to free beta1 or beta2-subunits without further modification of receptor structure. Functional studies performed in COS-7 cells showed that both mutants display an increased basal activation of the cAMP pathway when compared with the wild-type receptor. By contrast, deletion of almost the entire extracellular domain of the receptor (TM409 mutant) totally impairs receptor function, thus confirming a role of the juxtamembrane extracellular region in receptor function. The beta1 mutant receptor exhibited an increased internalization when compared with the hormone-activated holoreceptor. Furthermore, no recycling was observed in the case of the beta1 mutant receptor. These observations strongly argue for a different conformation between the receptor activated by cleavage and shedding on the one hand, and the receptor activated by the ligand on the other hand. Cleavage and shedding of a receptor already activated by a transmembrane activating mutation M453T further increase its activity, showing that the extracellular domain still exerts a negative effect in the M453T holoreceptor. An increased internalization of the M453T receptor was observed when compared with the wild-type receptor, which was increased further in the corresponding truncated beta1-M453T receptor. Thus cleavage and shedding yield TSHR activation but also increase internalization of the free beta-subunits of the receptor, the latter mechanism limiting simultaneously excessive receptor signaling. The combined effects may be responsible for the limited basal constitutive activation of the cAMP pathway that is detected for the TSHR.     

Properties of immobilized fig alpha-galactosidase and effect on ceramide-3 content of plasma from patients with Fabry's disease.

The possibility of lowering the level of ceramide-3 (galactosyl-alpha(1 leads to 4)-galactosyl-beta(1 leads to 4)-glucosyl-beta(1 leads to 1)-ceramide) in the plasma of patients with Fabry's disease was investigated. An immobilized alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was prepared by coupling purified fig alpha-galactosidase to Sepharose 4B. The pH optimum for the hydrolysis of the artificial substrate p-nitro-phenyl-alpha-D-galactopyranoside was shifted by approx. 0.5--1.0 pH unit to higher pH values upon coupling of the enzyme to Sepharose 4B. The immobilized enzyme was more stable than the native enzyme to incubation at 60 degrees C. The immobilized enzyme was able to hydrolyse ceramide-3 either at pH 4.5 or at pH 7.4 in an artificial system in which sodium taurocholate was used to solubilize the substrate. In contrast, when the immobilized enzyme was incubated with normal plasma or plasma from a patient with Fabry's disease, in which elevated levels of ceramide-3 occur, no hydrolysis of the glycosphingo-lipid could be detected. The results suggest that lowering of level of ceramide-3 in plasma from patients with Fabry's disease by enzymic means is not feasible.     

Characterization of two new glycosyl hydrolases from the lactic acid bacterium Carnobacterium piscicola strain BA.

Three genes with homology to glycosyl hydrolases were detected on a DNA fragment cloned from a psychrophilic lactic acid bacterium isolate, Carnobacterium piscicola strain BA. A 2.2-kb region corresponding to an alpha-galactosidase gene, agaA, was followed by two genes in the same orientation, bgaB, encoding a 2-kb beta-galactosidase, and bgaC, encoding a structurally distinct 1.76-kb beta-galactosidase. This gene arrangement had not been observed in other lactic acid bacteria, including Lactococcus lactis, for which the genome sequence is known. To determine if these sequences encoded enzymes with alpha- and beta-galactosidase activities, we subcloned the genes and examined the enzyme properties. The alpha-galactosidase, AgaA, hydrolyzes para-nitrophenyl-alpha-D-galactopyranoside and has optimal activity at 32 to 37 degrees C. The beta-galactosidase, BgaC, has an optimal activity at 40 degrees C and a half-life of 15 min at 45 degrees C. The regulation of these enzymes was tested in C. piscicola strain BA and activity on both alpha- and beta-galactoside substrates decreased for cells grown with added glucose or lactose. Instead, an increase in activity on a phosphorylated beta-galactoside substrate was found for the cells supplemented with lactose, suggesting that a phospho-galactosidase functions during lactose utilization. Thus, the two beta-galactosidases may act synergistically with the alpha-galactosidase to degrade other polysaccharides available in the environment.     

A case of Fabry's disease in a patient with no alpha-galactosidase A activity caused by a single amino acid substitution of Pro-40 by Ser

We analyzed a male patient with Fabry's disease who had no activity of the lysosomal hydrolase alpha-galactosidase A (alpha-GalA) and female members of his family. We cloned a cDNA that encoded the mutant alpha-GalA, determined its nucleotide sequence, and found two nucleotide differences between the mutant and the wild-type cDNAs. Although one difference was silent, the other difference, a C-to-T transition at nucleotide number 118, resulted in an amino acid substitution of Pro-40 by Ser. A transient expression assay demonstrated that this missense mutation was the cause of the deficiency of alpha-GalA activity in the patient. In vitro mutagenesis experiments demonstrated that Pro-40 is critical for the appearance of alpha-GalA activity.     

alpha-galactosidase A from human placenta. Stability and subunit size.

alpha-Galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was purified from human placenta. The purified enzyme showed one major band on polyacrylamide gel electrophoresis and a single precipitin line on double immunodiffusion. Electrophoresis of the purified, S-carboxymethylated enzyme on sodium dodecyl sulfate polyacrylamide gel showed one component with a molecular weight of about 65 000, but electrophoresis of the non-S-carboxymethylated enzyme showed two components, a major band with a molecular weight of 67 500 and a diffuse band with a molecular weight of 47 000. We suggest that the smaller diffuse component is a degradation product and that the enzyme is a dimer with a molecular weight of approximately 150 000 and a subunit of molecular weight of about 67 500. Antibody raised against the purified enzyme quantitatively precipitated alpha-galactosidase A, but not alpha-galactosidase in Fabry's disease fibroblasts. The alpha-galactosidase A is very heat labile and pH sensitive. It is most stable in concentrated solution at low temperature and at a pH of 5.0 to 6.0. When added to plasma at 37 degrees C, it has a half-life of only 17 min. This imposes a serious obstacle to its use in the treatment of Fabry's disease.     

Effect of tyrosine kinase inhibitors on clathrin-coated pit recruitment and internalization of epidermal growth factor receptor

Several inhibitors of epidermal growth factor receptor (EGFR) kinase and Src family kinases (SFK) were employed to study the role of these kinases in EGFR internalization through clathrin-coated pits. The EGFR kinase-specific compound PD158780 substantially diminished EGFR internalization. PP2, an inhibitor of SFK, had a moderate effect on EGFR internalization in several types of cells, including cells lacking SFK, indicating that the inhibition of endocytosis by PP2 is mediated by kinases other than SFK. In contrast, SU6656, a more specific inhibitor of SFK, did not affect EGFR internalization. To examine what stage of internalization requires receptor kinase activity, we established a quantitative assay based on three-dimensional fluorescence microscopy that measures co-localization of an EGF-rhodamine conjugate and a fluorescently tagged clathrin adaptor protein complex, AP-2. Interestingly, recruitment of EGFR into coated pits did not require physiological temperature because the maximal accumulation of EGFR in coated pits was observed at 4 degrees C. Pretreatment of the cells with PD158780 prevented EGFR recruitment into coated pits, whereas the inhibitor did not block the internalization of receptors that had first been allowed to enter the coated pits at 4 degrees C. These data demonstrate that the activation of receptor kinase is essential for the initial, coated pit recruitment step of endocytosis.     

Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugate.

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent internalization upon activation. The parathyroid hormone (PTH) receptor undergoes endocytosis following prolonged exposure to ligand although the ultimate fate of the receptor following internalization is largely unknown. To investigate compartmentalization of the PTH receptor, we have established a stable cell line expressing a PTH receptor-green fluorescent protein (PTHR-GFP) conjugate and an algorithm to quantify PTH receptor internalization. HEK 293 cells expressing the PTHR-GFP were compared with cells expressing the wild-type PTH receptor in whole-cell binding and functional assays. 125I-PTH binding studies revealed similar Bmax and kD values in cells expressing either the PTHR-GFP or the wild-type PTH receptor. PTH-induced cAMP accumulation was similar in both cell lines suggesting that addition of the GFP to the cytoplasmic tail of the PTH receptor does not alter the ligand binding or G-protein coupling properties of the receptor. Using confocal fluorescence microscopy, we demonstrated that PTH treatment of cells expressing the PTHR-GFP conjugate produced a time-dependent redistribution of the receptor to the endosomal compartment which was blocked by pretreatment with PTH antagonist peptides. Treatment with hypertonic sucrose prevented PTH-induced receptor internalization, suggesting that the PTH receptor internalizes via a clathrin-dependent mechanism. Moreover, co-localization with internalized transferrin showed that PTHR-GFP trafficking utilized the endocytic recycling compartment. Experiments using cycloheximide to inhibit protein synthesis demonstrated that recycling of the PTHR-GFP back to the plasma membrane was complete within 1-2 h of ligand removal and was partially blocked by pretreatment with cytochalasin D, but not nocodazole. We also demonstrated that the PTH receptor, upon recycling to the plasma membrane, is capable of undergoing a second round of internalization, a finding consistent with a role for receptor recycling in functional resensitization.     

Quantification of G-Protein Coupled Receptor Internatilization Using G-Protein Coupled Receptor-Green Fluorescent Protein Conjugates with the ArrayScantrade mark High-Content Screening System

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent homologous desensitization and internalization. Desensitization, defined as a decrease in the responsiveness to ligand, is accompanied by receptor aggregation on the cell surface and internalization via clathrin-coated pits to an intracellular endosomal compartment. In this study, we have taken advantage of the trafficking properties of GPCRs to develop a useful screening method for the identification of receptor mimetics. A series of studies were undertaken to evaluate the expression, functionality, and ligand-dependent trafficking of GPCR-green fluorescent protein (GFP) fusion conjugates stably transfected into HEK 293 cells. These GPCR-GFP expressing cells were then utilized in the validation of the ArrayScantrade mark (Cellomicstrade mark, Pittsburgh, PA), a microtiter plate imaging system that permits cellular and subcellular quantitation of fluorescence in whole cells. These studies demonstrated our ability to measure the internalization of a parathyroid hormone (PTH) receptor-GFP conjugate after ligand treatment by spatially resolving internalized receptors. Internalization was time- and dose-dependent and appeared to be selective for PTH. Similar results were obtained for a beta(2)-adrenergic receptor (beta(2) AR)-GFP conjugate stably expressed in HEK 293 cells. The internalized GFP-labeled receptors were visualized as numerous punctate 3spots2 within the cell interior. An algorithm has been developed that identifies and collects information about these spots, allowing quantification of the internalization process. Variables such as the receptor-GFP expression level, plating density, cell number per field, number of fields scanned per well, spot size, and spot intensity were evaluated during the development of this assay. The method represents a valuable tool to screen for receptor mimetics and antagonists of receptor internalization in whole cells rapidly.     

Serum concentration of alpha-2-macroglobulin, alpha-1-antitrypsin and alpha-2-antichymotrypsin in patients with lung carcinoma

Serum concentration of alpha-2-macroglobulin, alpha-1-antitrypsin and alpha-2-antichymotrypsin was evaluated in 26 patients with lung carcinoma. We observed an evident decrease in alpha-2-M and alpha-1-antitrypsin level and no differences between tested and control groups in alpha-1-antichymotrypsin concentration. The deficiency of protease inhibitors may be due to the increased level of protease activity in malignant cells. Infiltration of granulocytes near tumor and released enzymes from them may exhaust proteolytic inhibitory capacity, too. Increased protease activity is associated with transformation and uncontrolled proliferation, therefore antiproteases may be accepted as anticancerogenic factors. Further investigations are needed to bring us closer to understanding this question.     

Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase

Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA-). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA+ or PKA- cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA- cells. The m2 receptor was more sensitive to agonist-dependent internalization, and its rate of internalization was faster in PKA- cells than it was in PKA+ cells. Treatment of PKA+ cells with 8-(4-chlorophenylthio)-cAMP or forskolin did not result in internalization of either m1 or m2 receptors and did not alter the extent of agonist-dependent internalization of m2. These data indicate that the basal activity of PKA may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA+ and PKA- cells was accompanied by desensitization of functional responses. Exposure of PKA+ cells to 10(-7) M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 +/- 9% decrease in the number of m1 receptors on the cell surface. However, treatment of PKA- cells expressing the m1 receptor did not result in internalization, suggesting that PKA was required for some aspect of PMA-dependent internalization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of alpha-galactosidase digestion on lectin staining in human pancreas

Effects of alpha-galactosidase (from green coffee beans) digestion on lectin staining were examined in formalin-fixed, paraffin-embedded human pancreatic tissues from individuals of blood-group B and AB. Digestion with the enzyme resulted in almost complete loss of Griffonia simplicifolia agglutinin I-B4 (GSAI-B4) staining in the acinar cells with concomitant appearance of Ulex europaeus agglutinin-I(UEA-I) staining in the corresponding cells. In addition, reactivity with soybean agglutinin(SBA) was also imparted by the enzyme digestion in GSAI-B4 positive acinar cells. beta-Galactosidase digestion following alpha-galactosidase digestion neither reduced the reactivity with SBA nor induced the reactivity with Griffonia simplicifolia agglutinin-II(GSA-II) in GSAI-B4 positive cells, while in UEA-I positive cells, both reduction of SBA reactivity and appearance of GSA-II reactivity occurred after simple beta-galactosidase digestion as well as sequential digestion with alpha- and beta-galactosidase. However, when alpha-L-fucosidase digestion procedure was inserted between alpha- and beta-galactosidase digestion, UEA-I staining imparted by alpha-galactosidase digestion was markedly decreased in intensity and GSA-II reactivity was appeared in GSAI-B4 positive acinar cells. Furthermore, after sequential digestion with alpha-galactosidase and fucosidase, reactivity with peanut agglutinin(PNA) was revealed in GSAI-B4 positive acinar cells as well as UEA-I positive cells in secretors. In non-secretors, strong PNA staining was usually observed in the acinar cells throughout the glands without enzyme digestion.(ABSTRACT TRUNCATED AT 250 WORDS)     

RACK1 associates with muscarinic receptors and regulates M(2) receptor trafficking

Receptor internalization from the cell surface occurs through several mechanisms. Some of these mechanisms, such as clathrin coated pits, are well understood. The M(2) muscarinic acetylcholine receptor undergoes internalization via a poorly-defined clathrin-independent mechanism. We used isotope coded affinity tagging and mass spectrometry to identify the scaffolding protein, receptor for activated C kinase (RACK1) as a protein enriched in M(2)-immunoprecipitates from M(2)-expressing cells over those of non-M(2) expressing cells. Treatment of cells with the agonist carbachol disrupted the interaction of RACK1 with M(2). We further found that RACK1 overexpression inhibits the internalization and subsequent down regulation of the M(2) receptor in a receptor subtype-specific manner. Decreased RACK1 expression increases the rate of agonist internalization of the M(2) receptor, but decreases the extent of subsequent down-regulation. These results suggest that RACK1 may both interfere with agonist-induced sequestration and be required for subsequent targeting of internalized M(2) receptors to the degradative pathway.     

Metabolism of alpha macroglobulins of rabbits: study of their half-life]

Rabbit alpha-1-M and alpha-2-M labelling was carried out in vitro with 131I and in vivo with 75-Seleno-methionine in order to determine the half-life of these proteins. alpha-2-M catabolism is faster than the alpha-1-M one. This result is the same when these proteins were obtained from a plasma of a rabbit exhibiting an inflammatory reaction though their half life was shorter.     

The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent process

The extracellular N terminus of the endothelin B (ET(B)) receptor is susceptible to limited proteolysis (cleavage at R64 downward arrow S65), but the regulation and the functional consequences of the proteolysis remain elusive. We analyzed the ET(B) receptor or an ET(B)-GFP fusion protein stably or transiently expressed in HEK293 cells. After incubation of cells at 4 degrees C, only the full-length ET(B) receptor was detected at the cell surface. However, when cells were incubated at 37 degrees C, N-terminal cleavage was observed, provided endothelin 1 was present during the incubation. Cleavage was not inhibited by internalization inhibitors (sucrose, phenylarsine oxide). However, in cells incubated with both internalization inhibitors and metalloprotease inhibitors (batimastat, inhibitor of TNFalpha-convertase) or metal chelators (EDTA, phenanthroline), the cleavage was blocked, indicating that metalloproteases cleave the agonist-occupied ET(B) receptor at the cell surface. Functional analysis of a mutant ET(B) receptor lacking the first 64 amino acids ([Delta2-64]ET(B) receptor) revealed normal functional properties, but a 15-fold reduced cell surface expression. The results suggest a role of the N-terminal proteolysis in the regulation of cell surface expression of the ET(B) receptor. This is the first example of a multispanning membrane protein, which is cleaved by a metalloprotease, but retains its functional activity and overall structure.     

Internalization of the cytotoxic molecules of T101 F(ab')2-(ricin-A-chain) immunotoxin into human T-leukemic cells

We have investigated the internalization step of an immunotoxin and its relationship with cytotoxicity, with the F(ab')2-T101(ricin-A-chain) immunotoxin, directed against the CD5 antigen expressed on leukemic CEM cells. We first demonstrated that the biological action of the conjugate was related to its entry into the cell by an energy-dependent endocytotic process. We also found that during the first hours of cell intoxication, internalization is not the rate-limiting step of immunotoxin cytotoxicity. Internalization becomes limiting in cell intoxication only when the entry rate is low. Lastly we show that ammonium chloride, which strongly enhances immunotoxin potency, acts on internalized molecules for a very short time, suggesting that this enhancer affects an early intracellular step.     

Cellular distribution of constitutively active mutant parathyroid hormone (PTH)/PTH-related protein receptors and regulation of cyclic adenosine 3',5'-monophosphate signaling by beta-arrestin2

PTH promotes endocytosis of human PTH receptor 1 (PTH1Rc) by activating protein kinase C and recruiting beta-arrestin2. We examined the role of beta-arrestin2 in regulating the cellular distribution and cAMP signaling of two constitutively active PTH1Rc mutants, H223R and T410P. Overexpression of a beta-arrestin2-green fluorescent protein (GFP) conjugate in COS-7 cells inhibited constitutive cAMP accumulation by H223R and T410P in a dose-dependent manner, as well as the response to PTH of both mutant and wild-type PTH1Rcs. The cellular distribution of PTH1Rc-GFP conjugates, fluorescent ligands, and ssarrestin2-GFP was analyzed by fluorescence microscopy in HEK-293T cells. In cells expressing either receptor mutant, a ligand-independent mobilization of beta-arrestin2 to the cell membrane was observed. In the absence of ligand, H223R and wild-type PTH1Rcs were mainly localized on the cell membrane, whereas intracellular trafficking of T410P was also observed. While agonists promoted beta-arrestin2-mediated endocytosis of bot PTH1Rc mutants, antagonists were rapidly internalized only with T410P. The protein kinases inhibitor, staurosporine, significantly decreased internalization of ligand-PTH1Rc mutant complexes, although the recruitment of beta-arrestin2 to the cell membrane was unaffected. Moreover, in cells expressing a truncated wild-type PTH1Rc lacking the C-terminal cytoplasmic domain, agonists stimulated translocation of beta-arrestin2 to the cell membrane followed by ligand-receptor complex internalization without associated beta-arrestin2. In conclusion, cAMP signaling by constitutively active mutant and wild-type PTH1Rcs is inhibited by a receptor interaction with beta-arrestin2 on the cell membrane, possibly leading to uncoupling from G(s)alpha. This phenomenon is independent from protein kinases activity and the receptor C-terminal cytoplasmic domain. In addition, there are differences in the cellular localization and internalization features of constitutively active PTH1Rc mutants H223R and T410P.