Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “internalization”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,639 records · Page 91Linked to original sources
PubMed2003

Patency and caliber of the internal jugular vein after neck dissection.

OBJECTIVE: To determine the incidence of thrombosis and change in caliber of the internal jugular vein after neck dissection. PATIENTS AND METHODS: Between September 1999 and December 2000, we studied 68 patients who underwent 76 neck dissection that spared the internal jugular vein. Thrombosis of the internal jugular vein was determined using duplex Doppler scans. The examination was performed about 10 days after the operation. Preoperative and postoperative imaging was available for 28 patients who underwent 32 neck dissections after April 2000. These 28 patients were divided into three groups. Group 1 consisted of 5 internal jugular veins which were treated with supraomohyoid neck dissection. Group 2 included 17 internal jugular veins which were treated with functional neck dissection. For group 3, ten patients underwent simultaneous bilateral neck dissections that spared the ipsilateral internal jugular vein with concurrent contralateral radical neck dissection. For each group, the mean ratio of the post- to pre-operative caliber of the internal jugular vein was calculated. Measurements were planned for 7 days, 1 month, and 3 months postoperatively. RESULTS: The overall patency of the 76 internal jugular veins after neck dissection was 100%. In all groups, the mean ratio of the post- to pre-operative caliber of the internal jugular vein was lowest during the early postoperative period, and gradually increased within 3 months after surgery. CONCLUSION: The caliber of the internal jugular veins decreased the most during the early postoperative period; however, internal jugular vein thrombosis is uncommon.

Aged↗
PubMed1997

Left atrial chamber and appendage function after internal atrial defibrillation: a prospective and serial transesophageal echocardiographic study.

OBJECTIVES: The purpose of this prospective study was to assess left atrial chamber and appendage function after internal atrial defibrillation of atrial fibrillation and to evaluate the time course of recovery. BACKGROUND: External cardioversion of atrial fibrillation may result in left atrial appendage dysfunction ("stunning") and may promote thrombus formation. In contrast to external cardioversion, internal atrial defibrillation utilizes lower energies; however, it is unknown whether the use of lower energies may avoid stunning of the left atrial appendage. METHODS: Transesophageal and transthoracic echocardiography were performed in 20 patients 24 h before and 1 and 7 days after internal atrial defibrillation to assess both left atrial chamber and appendage function. Transthoracic echocardiography was again performed 28 days after internal atrial defibrillation to assess left atrial function. The incidence and degree of spontaneous echo contrast accumulation (range 1+ to 4+) was noted, and peak emptying velocities of the left atrial appendage were measured before and after internal atrial defibrillation. To determine left atrial mechanical function, peak A wave velocities were obtained from transmitral flow velocity profiles. RESULTS: Sinus rhythm was restored in all patients. The mean +/- SD peak A wave velocities increased gradually after cardioversion, from 0.47 +/- 0.16 m/s at 24 h to 0.61 +/- 0.13 m/s after 7 days (p < 0.05) and 0.63 +/- 0.13 m/s after 4 weeks. Peak emptying velocities of the left atrial appendage were 0.37 +/- 0.16 m/s before internal atrial defibrillation, decreased significantly after internal atrial defibrillation to 0.23 +/- 0.1 m/s at 24 h (p < 0.01) and then recovered to 0.49 +/- 0.23 m/s (p < 0.01) after 7 days. The corresponding values for the degree of spontaneous echo contrast were 1.2 +/- 1.2 before internal atrial defibrillation versus 2.0 +/- 1.0 (p < 0.01) and 1.1 +/- 1.3 (p < 0.01) 1 and 7 days after cardioversion, respectively. One patient developed a new thrombus in the left atrial appendage, and another had a thromboembolic event after internal atrial defibrillation. CONCLUSIONS: Internal atrial defibrillation causes depressed left atrial chamber and appendage function and may result in the subacute accumulation of spontaneous echo contrast and development of new thrombi after cardioversion. These findings have important clinical implications for anticoagulation therapy before and after low energy internal atrial defibrillation in patients with atrial fibrillation.

Anticoagulants↗
PubMed2000

Natriuretic peptides stimulate cyclic guanosine monophosphate production in human saphenous vein and internal mammary artery.

OBJECTIVE: It has been shown previously that the internal mammary artery releases more cyclic guanosine monophosphate after stimulation with atrial natriuretic peptide than the saphenous vein, and that C-type natriuretic peptide possesses a cyclic guanosine monophosphate stimulating potential on saphenous vein bypass grafts. The present study was undertaken to investigate intracellular content and extracellular release of cyclic guanosine monophosphate, by the internal mammary artery and saphenous vein, after challenge with further members of the natriuretic peptide family. METHODS: Specimens of human internal mammary artery and saphenous vein from 29 patients were cut into segments and stimulated with 10(-6) M concentrations of atrial natriuretic peptide (internal mammary artery n=8, saphenous vein n=10), brain natriuretic peptide (internal mammary artery n=9, saphenous vein n=13), C-type natriuretic peptide (internal mammary artery n=12, saphenous vein n=15), and urodilatin (internal mammary artery n=8, saphenous vein n=12). Intracellular content and extracellular release of cyclic guanosine monophosphate were determined using an (125)I radioimmunoassay. RESULTS: The following median stimulated intracellular cyclic guanosine monophosphate concentrations were measured in the internal mammary artery and saphenous vein: 35358 and 8672 fmol/cm(2) (P<0.001) after atrial natriuretic peptide, 45632 and 7830 fmol/cm(2) (P=0.003) after brain natriuretic peptide, 10144 and 13216 fmol/cm(2) (P=NS for intergraft comparison) after C-type natriuretic peptide, and 20949 and 6690 fmol/cm(2) (P=0.001) after urodilatin. Stimulation with atrial natriuretic peptide, brain natriuretic peptide and urodilation also led to a significant increase of extracellular cyclic guanosine monophosphate release by the internal mammary artery. CONCLUSIONS We conclude that brain natriuretic peptide and urodilatin exhibit a similarly effective cyclic guanosine monophosphate-stimulating potential on the internal mammary artery as atrial natriuretic peptide. In contrast, C-type natriuretic peptide shows comparable effects on the internal mammary artery and saphenous vein.

Atrial Natriuretic Factor↗
PubMed2001

Internalization of the human N-formyl peptide and C5a chemoattractant receptors occurs via clathrin-independent mechanisms.

After stimulation by ligand, most G protein-coupled receptors (GPCRs) undergo rapid phosphorylation, followed by desensitization and internalization. In the case of the N-formyl peptide receptor (FPR), these latter two processing steps have been shown to be entirely dependent on phosphorylation of the receptor's carboxy terminus. We have previously demonstrated that FPR internalization can occur in the absence of receptor desensitization, indicating that FPR desensitization and internalization are regulated differentially. In this study, we have investigated whether human chemoattractant receptors internalize via clathrin-coated pits. Internalization of the FPR transiently expressed in HEK 293 cells was shown to be dependent upon receptor phosphorylation. Despite this, internalization of the FPR, as well as the C5a receptor, was demonstrated to be independent of the actions of arrestin, dynamin, and clathrin. In addition, we utilized fluorescence microscopy to visualize the FPR and beta(2)-adrenergic receptor as they internalized in the same cell, revealing distinct sites of internalization. Last, we found that a nonphosphorylatable mutant of the FPR, unable to internalize, was competent to activate p44/42 MAP kinase. Together, these results demonstrate not only that the FPR internalizes via an arrestin-, dynamin-, and clathrin-independent pathway but also that signal transduction to MAP kinases occurs in an internalization-independent manner.

Antigens, CD↗
PubMed1990

The rate of internalization of different receptor-ligand complexes in alveolar macrophages is receptor-specific.

To probe the mechanisms of endocytosis in alveolar macrophages, we examined the internalization rates of three different receptors. Initial rates of internalization for mannosylated BSA, diferric transferrin and alpha-macroglobulin-proteinase complexes were all different. Although the absolute rates of internalization varied depending on the cell preparation, transferrin was internalized at 10-20% and alpha-macroglobulin-proteinase complex at 40-60% of the rate of manosylated-BSA. Incubation of cells with transferrin did not affect the rate of internalization of mannosylated BSA or alpha-macroglobulin-proteinase complexes, and the rates of internalization were independent of receptor occupancy. These different internalization rates could not be ascribed to different rates of diacytosis. Altering the distribution of unoccupied surface receptors by either trypsin treatment of cells at 0 degree C or exposure to hyperosmotic solutions resulted in the absolute internalization rates being affected by the experimental condition, but the hierarchy in receptor internalization rates was maintained. The fact that a variety of conditions affect receptor internalization rates to the same degree implies the existence of co-ordinate regulation at a single rate-limiting step. Based on these results, we suggest that differences in internalization rate reflect the ability of ligand-receptor complexes to be captured by coated pits.

Animals↗
PubMed2003

Receptor internalization is required for eotaxin-induced responses in human eosinophils.

BACKGROUND: CC chemokine receptor 3 (CCR3) is a major chemokine receptor involved in regulating eosinophil trafficking, and therefore the elucidation of ligand-induced CCR3 events has important implications in understanding the biologic and pathologic properties of eosinophils. After ligand binding to CCR3, cellular signals include stimulatory (ie, calcium mobilization, actin polymerization, shape change, and chemotaxis) and inhibitory (ie, desensitization of the receptor) events. We have previously demonstrated that CCR3 undergoes rapid and prolonged ligand-induced internalization. OBJECTIVE: Here we explore the role of internalization in downstream cellular processes, including shape change, actin polymerization, calcium mobilization, and desensitization. METHODS: Peripheral blood-derived human eosinophils were pretreated with 2 mechanistically distinct inhibitors of internalization, sucrose and phenylarsine oxide, and functional responses were monitored. RESULTS: We first demonstrate that ligand-induced internalization is required for chemokine-induced eosinophil shape change. To define which signaling components upstream of eosinophil shape change required internalization, we next studied the role of internalization in calcium mobilization and actin polymerization. Sucrose and phenylarsine oxide pretreatment inhibited actin polymerization, implicating receptor internalization in this early response. In contrast, calcium mobilization was not inhibited by blockade of internalization. Finally, we were interested in testing the role of internalization in receptor desensitization. We first demonstrated that preincubation with eotaxin induced a dose-dependent desensitization in eotaxin-induced eosinophil transepithelial migration. However, this phenomenon was not inhibited by blockade of internalization. CONCLUSION: These results establish that CCR3 internalization is critically involved in select eosinophil functional responses (ie, cellular shape change and actin polymerization) but not desensitization and calcium mobilization.

Eosinophils↗
PubMed2001

Chymase-dependent angiotensin II formation in the saphenous vein versus the internal thoracic artery.

OBJECTIVES: The great saphenous vein graft is known to be less patent than the internal thoracic artery graft. Recently, we reported that chymase-dependent angiotensin II formation plays an important role in the development of intimal hyperplasia in dog grafted veins. In this study we investigated the levels of angiotensin II-forming enzymes, angiotensin-converting enzyme, and chymase in human saphenous veins and internal thoracic arteries. METHODS: The saphenous vein and internal thoracic artery specimens were obtained from coronary artery bypass grafts of patients during surgical procedures (saphenous vein, n = 16; internal thoracic artery, n = 16). Activities of angiotensin-converting enzyme and chymase were determined by using the extract from the saphenous vein or internal thoracic artery. Sections of the saphenous vein or internal thoracic artery were stained with van Gieson's elastin stain and were immunostained with anti-human chymase antibody. RESULTS: The activities of angiotensin-converting enzyme in the saphenous vein and internal thoracic artery were 0.34 +/- 0.12 and 0.32 +/- 0.17 mU/mg protein, respectively, and the difference was not significant. The chymase activity in the saphenous vein was significantly higher than that in the internal thoracic artery (saphenous vein, 10.1 +/- 0.81 mU/mg protein; internal thoracic artery, 6.21 +/- 1.86 mU/mg protein). Chymase-positive cells in the saphenous vein were located in both the media and adventitia, and those in the internal thoracic artery were located only in the adventitia. The number of chymase-positive cells in the saphenous vein was about 2.6 times that in the internal thoracic artery. CONCLUSION: The chymase activity, but not the angiotensin-converting enzyme activity, was significantly higher in the saphenous vein, suggesting that the high levels of chymase activity may be related to the poorer performance of the saphenous vein for use as a bypass conduit.

Aged↗
PubMed1995

Dynamic or stable interactions of influenza hemagglutinin mutants with coated pits. Dependence on the internalization signal but not on aggregation.

Measurements of the lateral mobility of native and mutated membrane proteins, combined with treatments that alter clathrin lattice structure, are capable of characterizing their interactions with coated pits in live cells (Fire, E., Zwart, D. E., Roth, M. G., and Henis, Y. I. (1991) J. Cell Biol. 115, 1585-1594). To explore the dependence of these interactions on the internalization signal and the aggregation state of the protein, we have extended this approach to investigate the interactions between coated pits and several influenza hemagglutinin (HA) mutants, which differ in the internalization signals in their short cytoplasmic tails. The lack of internalization signals in the trimeric wild-type HA enables a direct comparison between specific internalization signals introduced singly in each mutant. We have selected for these studies HA mutants that showed different internalization rates and varied in their tendency to aggregate into complexes larger than trimers. Our results indicate that the mode of interaction with coated pits (transient association-dissociation versus stable entrapment) depends on the internalization signal and affects the internalization efficiency. Mutants that contain a strong internalization signal and undergo fast endocytosis were entrapped in coated pits for the entire duration of the lateral mobility measurement, suggesting stable association with (slow dissociation from) coated pits. A mutant with a suboptimal internalization signal, which was internalized 10-fold slower, exhibited transient interactions with coated pits. Both types of interactions disappeared or were significantly reduced upon disruption of the clathrin lattices under hypertonic conditions, and were modulated following the "freezing" of coated pits by cytosol acidification. Unlike the dependence on the cytoplasmic internalization signal, the interactions with coated pits did not depend on the aggregation state (measured by sucrose gradient centrifugation after solubilization in n-octylglucoside) of the mutants.

Amino Acid Sequence↗
PubMed1999

Trafficking of the HIV coreceptor CXCR4. Role of arrestins and identification of residues in the c-terminal tail that mediate receptor internalization.

The G protein-coupled chemokine receptor CXCR4 serves as the primary coreceptor for entry of T-cell tropic human immunodeficiency virus. CXCR4 undergoes tonic internalization as well as internalization in response to stimulation with phorbol esters and ligand (SDF-1alpha). We investigated the trafficking of this receptor, and we attempted to define the residues of CXCR4 that were critical for receptor internalization. In both COS-1 and HEK-293 cells transiently overexpressing CXCR4, SDF-1alpha and phorbol esters (PMA) promoted rapid internalization of cell surface receptors as assessed by both enzyme-linked immunosorbent assay and immunofluorescence analysis. Expression of GRK2 and/or arrestins promoted modest additional CXCR4 internalization in response to both PMA and SDF. Both PMA- and SDF-mediated CXCR4 internalization was inhibited by coexpression of dominant negative mutants of dynamin-1 and arrestin-3. Arrestin was also recruited to the plasma membrane and appeared to colocalize with internalized receptors in response to SDF but not PMA. We then evaluated the ability of CXCR4 receptors containing mutations of serines and threonines, as well as a dileucine motif, within the C-terminal tail to be internalized and phosphorylated in response to either PMA or SDF-1alpha. This analysis showed that multiple residues within the CXCR4 C-terminal tail appear to mediate both PMA- and SDF-1alpha-mediated receptor internalization. The ability of coexpressed GRK2 and arrestins to promote internalization of the CXCR4 mutants revealed distinct differences between respective mutants and suggested that the integrity of the dileucine motif (Ile-328 and Leu-329) and serines 324, 325, 338, and 339 are critical for receptor internalization.

Amino Acid Sequence↗
PubMed2000

Multiple topological domains mediate subtype-specific internalization of the M2 muscarinic acetylcholine receptor.

Endocytosis of agonist-activated G protein-coupled receptors (GPCRs) is required for both resensitization and recycling to the cell surface as well as lysosomal degradation. Thus, this process is crucial for regulation of receptor signaling and cellular responsiveness. Although many GPCRs internalize into clathrin-coated vesicles in a dynamin-dependent manner, some receptors, including the M(2) muscarinic acetylcholine receptor (mAChR), can also exhibit dynamin-independent internalization. We have identified five amino acids, located in the sixth and seventh transmembrane domains and the third intracellular loop, that are essential for agonist-induced M(2) mAChR internalization via a dynamin-independent mechanism in JEG-3 choriocarcinoma cells. Substitution of these residues into the M(1) mAChR, which does not internalize in these cells, is sufficient for conversion to the internalization-competent M(2) mAChR phenotype, whereas removal of these residues from the M(2) mAChR blocks internalization. Cotransfection of a dominant-negative isoform of dynamin has no effect on M(2) mAChR internalization. An internalization-incompetent M(2) mutant that lacks a subset of the necessary residues can still internalize via a G protein-coupled receptor kinase-2 and beta-arrestin-dependent pathway. Furthermore, internalization is independent of the signal transduction pathway that is activated. These results identify a novel motif that specifies structural requirements for subtype-specific dynamin-independent internalization of a GPCR.

Amino Acid Sequence↗
PubMed2003

Galphaq-coupled receptor internalization specifically induced by Galphaq signaling. Regulation by EBP50.

In the present report, we investigated the effect of ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) expression on the agonist-induced internalization of the thromboxane A(2) beta receptor (TPbeta receptor). Interestingly, we found that EBP50 almost completely blocked TPbeta receptor internalization, which could not be reversed by overexpression of G protein-coupled receptor (GPCR) kinases and arrestins. Because we recently demonstrated that EBP50 can bind to and inhibit Galpha(q), we next studied whether Galpha(q) signaling could induce TPbeta receptor internalization, addressing the long standing question about the relationship between GPCR signaling and their internalization. Expression of a constitutively active Galpha(q) mutant (Galpha(q)-R183C) resulted in a robust internalization of the TPbeta receptor, which was unaffected by expression of dominant negative mutants of arrestin-2 and -3, but inhibited by expression of EBP50 or dynamin-K44A, a dominant negative mutant of dynamin. Phospholipase Cbeta and protein kinase C did not appear to significantly contribute to internalization of the TPbeta receptor, suggesting that Galpha(q) induces receptor internalization through a phospholipase Cbeta- and protein kinase C-independent pathway. Surprisingly, there appears to be specificity in Galpha protein-mediated GPCR internalization. Galpha(q)-R183C also induced the internalization of CXCR4 (Galpha(q)-coupled), whereas it failed to do so for the beta(2)-adrenergic receptor (Galpha(s)-coupled). Moreover, Galpha(s)-R201C, a constitutively active form of Galpha(s), had no effect on internalization of the TPbeta, CXCR4, and beta(2)-adrenergic receptors. Thus, we showed that Galpha protein signaling can lead to internalization of GPCRs, with specificity in both the Galpha proteins and GPCRs that are involved. Furthermore, a new function has been described for EBP50 in its capacity to inhibit receptor endocytosis.

Arrestins↗
PubMed2004

A tyrosine-based sorting signal regulates intracellular trafficking of protease-activated receptor-1: multiple regulatory mechanisms for agonist-induced G protein-coupled receptor internalization.

Protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is irreversibly proteolytically activated, internalized, and then sorted to lysosomes and degraded. Internalization and lysosomal sorting of activated PAR1 is critical for termination of receptor signaling. We previously demonstrated that activated PAR1 is rapidly phosphorylated and internalized via a clathrin- and dynamin-dependent pathway that is independent of arrestins. Toward understanding the mechanisms responsible for activated PAR1 internalization through clathrin-coated pits we examined the function of a highly conserved tyrosine-based motif, YXXL, localized in the cytoplasmic carboxyl tail of the receptor. A mutant PAR1 in which tyrosine 383 and leucine 386 were replaced with alanines (Y383A/L386A) was significantly impaired in agonist-triggered internalization and degradation compared with wild-type receptor. In contrast, constitutive internalization, and recycling of unactivated PAR1 Y383A/L386A mutant was not affected, suggesting that tonic cycling of the mutant receptor remained intact. Strikingly, a PAR1 C387Z truncation mutant in which the YXXL motif was exposed at the C terminus constitutively internalized and degraded in an agonist-independent manner, whereas C387Z truncation mutant in which the critical tyrosine and leucine were mutated to alanine (C387Z-Y383A/L386A) failed to internalize. Inhibition of PAR1 C387Z mutant constitutive internalization with dominant-negative K44A dynamin blocked agonist-independent degradation of the mutant receptor. Together these findings strongly suggest that internalization of activated PAR1 is controlled by multiple regulatory mechanisms involving phosphorylation and a highly conserved tyrosine-based motif, YXXL. This study is the first to describe a function for a tyrosine-based motif, YXX, in GPCR internalization and reveal novel complexities in the regulation of GPCR trafficking.

Alanine↗
PubMed1995

Apical and basolateral coated pits of MDCK cells differ in their rates of maturation into coated vesicles, but not in the ability to distinguish between mutant hemagglutinin proteins with different internalization signals.

In polarized epithelial MDCK cells, all known endogenous endocytic receptors are found on the basolateral domain. The influenza virus hemagglutinin (HA) which is normally sorted to the apical plasma membrane, can be converted to a basolateral protein by specific mutations in its short cytoplasmic domain that also create internalization signals. For some of these mutations, sorting to the basolateral surface is incomplete, allowing internalization of two proteins that differ by a single amino acid of the internalization signal to be compared at both the apical and basolateral surfaces of MDCK cells. The rates of internalization of HA-Y543 and HA-Y543,R546 from the basolateral surface of polarized MDCK cells resembled those in nonpolarized cells, whereas their rates of internalization from the apical cell surface were fivefold slower. However, HA-Y543,R546 was internalized approximately threefold faster than HA-Y543 at both membrane domains, indicating that apical endocytic pits in polarized MDCK cells retained the ability to discriminate between different internalization signals. Slower internalization from the apical surface could not be explained by a limiting number of coated pits: apical membrane contained 0.7 as many coated pits per cell cross-section as did basolateral membranes. 10-14% of HA-Y543 at the apical surface of polarized MDCK cells was found in coated pits, a percentage not significantly different from that observed in apical coated pits of nonpolarized MDCK cells, where internalization was fivefold faster. Thus, there was no lack of binding sites for HA-Y543 in apical coated pits of polarized cells. However, at the apical surface many more shallow pits, and fewer deep, mature pits, were observed than were seen at the basolateral. These results suggest that the slower internalization at the apical surface is due to slower maturation of coated pits, and not to a difference in recognition of internalization signals.

Animals↗
PubMed2003

Pelvic examination training for interns: a randomized controlled trial.

PURPOSE: To evaluate the effectiveness of pelvic examination training for internal medicine interns conducted by instructors who also serve as models for the examination. METHOD: In 2001, 80 interns from three university internal medicine residencies completed questionnaires about their experiences with pelvic examinations. Interns who were available for training underwent baseline assessment of their pelvic examination skills and were randomized to training or to a control group that received only educational literature. Skills were assessed with a 29-item checklist at baseline and at follow-up by evaluators blinded to group assignment, and interrater agreement was estimated by review of audiotapes. RESULTS: Seventy-two interns were randomized and underwent baseline skills assessment. Seventy interns returned for follow-up assessment after approximately 14 weeks (range, 10-17 weeks). The 39 interns randomized to training and 33 randomized to the control group did not differ with respect to age, gender, or prior pelvic examination training. In both groups there was substantial variability in skills at baseline, with graduates of U.S. medical schools scoring significantly higher than did graduates of non-U.S. medical schools (0.66 versus 0.41, p < 10(-5)). Interns randomized to training had significantly higher scores at follow-up than did interns in the control group (0.79 versus 0.57, p < 10(-6)). For seven items verifiable by audiotape, interrater agreement was good (overall kappa, 0.54; range among items, 0.3-0.85). CONCLUSION: Specialized trainers can reliably evaluate and improve the pelvic examination skills of interns, and improvements are demonstrable three months after training. Further research is needed to ascertain whether training efficiency can be improved and to measure the impact of training on patient satisfaction and clinical outcomes.

Adult↗
PubMed2002

Differential internalization of the prostaglandin f(2alpha) receptor isoforms: role of protein kinase C and clathrin.

FP prostanoid receptors are G-protein-coupled receptors that mediate the actions of prostaglandin F(2alpha) (PGF(2alpha)). Alternative mRNA splicing gives rise to two isoforms, FP(A) and FP(B), which are identical except for their intracellular carboxyl termini. In this study, we examined the internalization of recombinant FLAG-epitope-tagged FP(A) and FP(B) receptors that were stably expressed in human embryonic kidney-293 cells. Cell surface receptors on live cells were labeled with anti-FLAG antibodies either in the presence or absence of PGF(2alpha) and were examined by immunofluorescence microscopy. In the absence of PGF(2alpha), FP(A)-expressing cells were labeled predominantly on the cell surface; however, FP(B)-expressing cells were labeled on both the cell surface and intracellularly, indicating constitutive internalization of the FP(B) isoform. After treatment with PGF(2alpha), FP(A)-expressing cells were labeled intracellularly, reflecting receptor internalization, which could be mimicked with phorbol 12-myristyl 13-acetate (PMA), an activator of protein kinase C (PKC). Pretreatment of FP(A)-expressing cells with Gö 6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo[2,3-a]pyrrolo[3,4-c]carbozole], an inhibitor of PKC, blocked both PGF(2alpha)- and PMA-induced receptor internalization. However, Gö 6976 did not block constitutive internalization of the FP(B) isoform, suggesting that the mechanisms of receptor internalization differ between the FP(A) and FP(B) isoforms. Furthermore, pretreatment with sucrose, an inhibitor of clathrin-dependent internalization, blocked PGF(2alpha)-induced internalization of the FP(A) isoform but did not block constitutive internalization of the FP(B) isoform. In conclusion, the FP(A) receptor isoform shows an agonist-induced internalization involving PKC and clathrin, whereas the FP(B) isoform undergoes agonist-independent internalization that does not involve PKC or clathrin.

Cell Line↗
PubMed1988

Internalization and cellular processing of cholecystokinin in rat pancreatic acinar cells.

To evaluate the internalization of cholecystokinin, monoiodinated imidoester of cholecystokinin octapeptide [125I-(IE)-CCK-8] was bound to dispersed pancreatic acinar cells, and surface-bound and internalized radioligand were differentiated by treating with an acidified glycine buffer. The amount of internalized radioligand was four- and sevenfold greater at 24 and 37 degrees C than at 4 degrees C between 5 and 60 min of association. Specific binding of radioligand to cell surface receptors was not significantly different at these temperatures. Chloroquine, a lysosomotropic agent that blocks intracellular proteolysis, significantly increased the amount of CCK-8 internalized by 18 and 16% at 30 and 60 min of binding, respectively, compared with control. Dithiothreitol (DTT), a sulfhydryl reducing agent, also augmented the amount of CCK-8 radioligand internalized by 25 and 29% at 30 and 60 min, respectively. The effect of chloroquine and DTT on the processing of internalized radioligand was also considered after an initial 60 min of binding of radioligand to acinar cells. After 180 min of processing, the amount of radioligand internalized was significantly greater in the presence of chloroquine compared with controls, whereas the amount of radioligand declined in acinar cells treated with DTT. Internalized and released radioactivity from acinar cells was rebound to pancreatic membrane homogenates to determine the amount of intact radioligand during intracellular processing. Chloroquine significantly increased the amount of intact 125I-(IE)-CCK-8 radioligand in released and internalized radioactivity while DTT increased the amount of intact radioligand only in internalized samples. This study shows that pancreatic acinar cells rapidly internalize large amounts of CCK-8 and that chloroquine and DTT inhibit intracellular degradation.

Animals↗
PubMed2003

Receptor activation and 2 distinct COOH-terminal motifs control G-CSF receptor distribution and internalization kinetics.

We have studied the intracellular distribution and internalization kinetics of the granulocyte colony-stimulating factor receptor (G-CSF-R) in living cells using fusion constructs of wild-type or mutant G-CSF-R and enhanced green fluorescent protein (EGFP). Under steady-state conditions the G-CSF-R localized predominantly to the Golgi apparatus, late endosomes, and lysosomes, with only low expression on the plasma membrane, resulting from spontaneous internalization. Internalization of the G-CSF-R was significantly accelerated by addition of G-CSF. This ligand-induced switch from slow to rapid internalization required the presence of G-CSF-R residue Trp650, previously shown to be essential for its signaling ability. Both spontaneous and ligand-induced internalization depended on 2 distinct amino acid stretches in the G-CSF-R COOH-terminus: 749-755, containing a dileucine internalization motif, and 756-769. Mutation of Ser749 at position -4 of the dileucine motif to Ala significantly reduced the rate of ligand-induced internalization. In contrast, mutation of Ser749 did not affect spontaneous G-CSF-R internalization, suggesting the involvement of a serine-threonine kinase specifically in ligand-accelerated internalization of the G-CSF-R. COOH-terminal truncation mutants of G-CSF-R, found in severe congenital neutropenia, lack the internalization motifs and were completely defective in both spontaneous and ligand-induced internalization. As a result, these mutants showed constitutively high cell-surface expression.

3T3 Cells↗
PubMed1995

Internalization of the radioiodinated somatostatin analog [125I-Tyr3]octreotide by mouse and human pituitary tumor cells: increase by unlabeled octreotide.

Recently, we developed a technique that allows the in vivo visualization in man of somatostatin receptor-positive neuroendocrine tumors after i.v. injection of [125I-Tyr3]octreotide or [111In-DTPA-D-Phe1]octreotide. Radiotherapy of such tumors using somatostatin analogs coupled to alpha- or beta-emitting radionuclides has been proposed as an application for radiolabeled somatostatin analogs. To develop this concept further, it is of importance to know whether the above-mentioned radiolabeled somatostatin analogs are internalized by the tumor cells, and whether it might be possible to manipulate the degree of internalization. In the present study we investigated the internalization of a stable somatostatin analog, [125I-Tyr3]octreotide, by mouse AtT20/D16V pituitary tumor cells and primary cultures of human GH-secreting pituitary tumor cells. Treatment of the cells with low pH was used to distinguish between membrane-bound (acid-releasable) and internalize (acid-resistant) radioligand. [125I-Tyr3]octreotide showed a time-dependent increasing accumulation in AtT20 cells; after 4 h of incubation, values up to 6-8% of the dose of radioligand added were obtained. Binding and internalization of [125I-Tyr3]octreotide were temperature dependent and inhibited by pertussis toxin. Inhibitors of lysosomal degradation did not increase the amount of internalized radioligand. After 4 h of incubation, 88% of the radioactivity present in the cells was still peptide bound, suggesting a low intracellular breakdown of this radioligand. Six of seven human GH-secreting adenoma cell cultures also internalized [125I-Tyr3]octreotide (variation between 0.24-4.98% of the dose radioligand added). Displacement of binding and internalization of [125I-Tyr3]octreotide by unlabeled octreotide showed a bell-shaped curve in AtT20 cells. At low concentrations (0.1 and 1 nM), binding and internalization were increased, whereas at higher concentrations, saturation occurred. In contrast to this, binding of [125I-Tyr3]octreotide to a broken cell preparation of AtT20 cells was displaced in a dose-dependent manner by unlabeled octreotide, with an IC50 of 0.1 nM. Similar observations were made in the human GH-secreting adenoma cell cultures. In conclusion, a high amount of [125I-Tyr3]octreotide is internalized in a specific-, time-, temperature-, and pertussis toxin-sensitive GTP-binding protein-dependent manner by mouse AtT20 and human GH-secreting pituitary tumor cells. In the presence of a low concentration of unlabeled octreotide, a rapid increase in the amount of [125I-Tyr3]octreotide internalized by AtT20 cells and by the majority of the human GH-secreting adenoma cell cultures was found.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoma↗