Regulation of chemoattractant receptor interaction with transducing proteins by organizational control in the plasma membrane of human neutrophils

Isolated purified plasma membrane domains from unstimulated human neutrophils were photoaffinity labeled with F-Met-Leu-Phe-N epsilon-(2- (p-azido-[125I]salicylamido)ethyl- 1,3'-dithiopropionyl)-Lys also referred to as FMLPL-SASD[125I]. Most of the photoaffinity-labeled N- formyl peptide receptors were found in light plasma membrane fraction (PM-L) which has been previously shown to be enriched in guanyl nucleotide binding proteins and the plasma membrane marker alkaline phosphatase (Jesaitis, A. J., G. M. Bokoch, J. O. Tolley, and R. A. Allen. 1988. J. Cell Biol. 107:921-928). Furthermore, the heavy plasma membrane fraction (PM-H), which is enriched in actin and fodrin, was depleted in receptors. Solubilization of PM-L and PM-H in divalent cation-free buffer containing octylglucoside and subsequent sedimentation at 180,000 g in detergent-containing sucrose gradients revealed two receptor forms. The major population, found in PM-L sedimented as a globular protein with an apparent sedimentation coefficient of 6-7S, while a minor fraction found in the PM-H fraction sedimented as a 4S particle. In addition, the 6-7S form could be converted to the 4S form by inclusion of guanosine 5'-O-(3- thiotriphosphate) (GTP gamma S) in the extraction buffer (ED50 = 10-30 nM). ATP was not effective at doses of up to 10 microM. In contrast, isolation and solubilization of receptors from desensitized cells (photoaffinity labeled after a 15 degrees C incubation with FMLPL- SASD[125I]) revealed that the majority of receptors (greater than 60- 90%), which are found in PM-H, sedimented as 4S particles. A minor fraction of receptors found in the PM-L sedimented as 6-7S species. The receptors in the PM-H fraction, however, were still capable of interacting with G-proteins, since addition of unlabeled PM-L membrane fraction as a G-protein source reconstituted a more rapidly sedimenting form showing sensitivity to GTP gamma S. These results suggest that receptors in unstimulated human neutrophils have a higher probability of interacting with G-proteins because they are in the light plasma membrane domain. The results also suggest that receptors that have been translocated to the heavy plasma membrane domain during the process of desensitization or response termination have a lower probability of interacting with G-protein. Since the latter receptors are still capable of forming G protein associations, then their lateral segregation would represent a mechanism of controlling of receptor G- protein interactions. This reorganization of the plasma membrane, therefore, may form the molecular basis for response termination or homologous desensitization in human neutrophils.     

Subcellular localization of Gi alpha in human neutrophils

Subcellular fractions were prepared from human neutrophils by sucrose density gradient centrifugation and analyzed for Gi-like proteins by pertussis toxin-catalyzed [32P]ADP-ribosylation and by immunoblotting with rabbit antiserum AS/6 which recognizes purified transducin and Gi, but not Gs or Go alpha-subunits. In resting cells, approximately equal to 60% of pertussis toxin substrate retrieved from the sucrose density gradient localized to the plasma membrane-enriched fraction, approximately equal to 35% to the specific granule-enriched fraction, and approximately equal to 5% to cytosol. The azurophil granule-enriched fraction did not contain pertussis toxin substrate. In contrast to plasma membrane, the specific granule-enriched fraction demonstrated increased AS/6 immunoreactivity of a approximately equal to 41-kDa protein relative to a approximately equal to 40-kDa protein. Within the specific granule-enriched fraction, the peak of pertussis toxin substrate detected immunochemically or by [32P]ADP-ribosylation sedimented at a lighter density (rho = 1.6 g/ml) than did lactoferrin (rho = 1.19 g/ml), suggesting that the intracellular compartment bearing pertussis toxin substrate may not be the lactoferrin containing specific granule, per se. Furthermore, in neutrophils exposed to 10(-8) M N-formylmethionylleucylphenylalanine, a weak degranulating stimulus (7% lactoferrin degranulation), there was a 31-42% decline in pertussus toxin-catalyzed [32P]ADP-ribosylation of approximately equal to 40-41-kDa proteins in the specific granule-enriched fraction accompanied by a near-quantitative increase in labeling of plasma membrane. The pool of intracellular formyl peptide receptors localized to the specific granule-enriched fraction appeared functionally coupled to a cosedimenting G-protein in experiments demonstrating modulation of high affinity N-formylmethionylleucyl[3H]phenylalanine binding by guanosine 5'-(3-O-thio)triphosphate or pertussis toxin. The data indicate that neutrophils contain a surface translocatable pool of intracellular G-protein sedimenting in the specific granule-enriched fraction and support the view that mobilization of intracellular G-protein represents a mechanism by which cells can regulate receptor activity.     

Characterization of the formyl peptide chemotactic receptor appearing at the phagocytic cell surface after exposure to phorbol myristate acetate

We examined the biochemistry and subcellular source of new formyl peptide chemotactic receptor appearing at the human neutrophil and differentiated HL-60 (d-HL-60) cell surface after stimulation with phorbol myristate acetate (PMA). Formyl peptide receptor was analyzed by affinity labeling with formyl-norleu-leu-phe-norleu-[125I]iodotyr-lys and ethylene glycol bis(succinimidyl succinate) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometric analysis of autoradiographs. PMA, a specific granule secretagogue, increases affinity labeling of formyl peptide receptors on the neutrophil surface by 100%, and on d-HL-60, which lack specific granule markers, by 20%. Papain treatment markedly reduces surface labeling of formyl peptide receptor in both neutrophils and d-HL-60, and results in the appearance of a lower m.w. membrane-bound receptor fragment. PMA stimulation of papain-treated cells increases uncleaved surface receptor on neutrophils by 400%, and on d-HL-60 by only 45%. This newly appearing receptor is the same apparent m.w. (55,000 to 75,000 for neutrophils; 62,000 to 80,000 for d-HL-60) and yields the same papain cleavage product (Mr, 31,000 for neutrophils; Mr, 29,000 for d-HL-60) as receptor on the surface of unstimulated cells. Formyl peptide receptor detected by affinity labeling in neutrophil specific granule-enriched subcellular fractions is identical to receptor found on the surface of unstimulated cells appearing as equal amounts of two isoelectric forms (isoelectric points, 5.8 and 6.2) at Mr 55,000 to 70,000. There is twice as much receptor present in the specific granule-enriched fraction per cell equivalent compared with plasma membrane. Azurophil granules contain trace amounts of receptor. Similar analysis of neutrophils treated with papain before subcellular fractionation shows that papain cleaved receptor fragment is detectable almost exclusively in the plasma membrane-enriched fraction. Most of the affinity-labeled formyl peptide receptor present in specific granule enriched fraction is present in membranes other than plasma membrane or Golgi membrane, because specific granule-enriched fraction contains only a small amount of plasma membrane marker and an amount of Golgi membrane marker equal to that found in plasma membrane-enriched fraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of low-density Triton X-100-insoluble plasma membrane microdomains in higher plants.

Low density Triton X-100-insoluble plasma membrane microdomains can be isolated from different mammalian cell types and are proposed to be involved in membrane trafficking, cell morphogenesis and signal transduction. Heterotrimeric G-proteins and their receptors are often associated with such domains, suggesting that these structures are involved in G-protein-coupled signaling. Here we report that detergent-insoluble plasma membrane microdomains also exist in higher plants and contain about 15% of membrane-bound heterotrimeric G-protein beta-subunit (Gbeta). Plasma membrane microdomains were isolated from tobacco leaves. They have low buoyant density relative to the surrounding plasma membrane, and are insoluble in Triton X-100 at 4 degrees C. Detergent-insoluble vesicles were examined by freeze-fracture electron microscopy. They have sizes in the range 100-400 nm, and often contain aggregated protein complexes. The majority of plasma membrane proteins cannot be detected in the Triton X-100-insoluble fraction, while few polypeptides are highly enriched. We identified six proteins with molecular masses of 22, 28, 35, 60, 67 and 94 kDa in detergent-insoluble fractions that are glycosylphosphatidylinositol (GPI)-anchored.     

Significance of glycosphingolipid fatty acid chain length on membrane microdomain-mediated signal transduction

Lactosylceramide (LacCer), a neutral glycosphingolipid, is abundantly expressed on human neutrophils, and specifically recognizes several pathogenic microorganisms. LacCer forms membrane microdomains coupled with the Src family kinase Lyn on the plasma membrane, and ligand binding to LacCer activates Lyn, resulting in neutrophil functions. In contrast, neutrophilic differentiated HL-60 cells do not have Lyn-associated LacCer-enriched microdomains and lack LacCer-mediated functions. In neutrophil plasma membranes, the very long fatty acid C24:0 and C24:1 chains are the main components of LacCer, whereas plasma membrane of D-HL-60 cells mainly includes C16-LacCer species. Here, we suggest that LacCer species containing very long fatty acid chains are indispensable for the association of Lyn with LacCer-enriched microdomains and LacCer-mediated functions.     

Fate of surface proteins of rabbit polymorphonuclear leukocytes during phagocytosis. I. Identification of surface proteins

To study the fate of external membrane proteins during phagocytosis, rabbit peritoneal neutrophils were labeled by enzymatic iodination. Iodine was incorporated into at least 13 proteins ranging in size from approximately 250,000 to 18,000 daltons as judged from autoradiography of gels after SDS-polyacrylamide gel electrophoresis of labeled cells. The major contractile proteins of neutrophils, actin and myosin, were not labeled when intact cells were iodinated but were labeled when homogenates of these cells were iodinated. Nine of the iodinated proteins were released by mild protease treatment of intact cells. A plasma membrane-rich fraction was isolated by density centrifugation. This fraction was enriched at least 10-fold for lactoperoxidase-labeled acid-insoluble proteins. It was enriched to the same extent for the presence of iodinated wheat germ agglutinin that had been bound to intact cells at 4 degrees C before homogenization. Analysis of SDS-polyacrylamide gel electrophoresis revealed that the proteins of this fraction were predominantly of high molecular weight. However, only 8 of the 13 proteins iodinated on intact cells were found in this fraction. The remaining five were enriched in a dense fraction containing nuclei, intact cells, and membranous vesicles, and may represent a specialized segment of the neutrophil cell surface.     

Copurification of rho protein and the rho-GDP dissociation inhibitor from bovine neutrophil cytosol. Effect of phosphoinositides on rho ADP-ribosylation by the C3 exoenzyme of Clostridium botulinum.

The substrate of the C3 exoenzyme from botulinum toxin is a protein which is particularly abundant in the cytosol of neutrophils [Stasia, M. J., Jouan, A., Bourmeyster, N., Boquet, P., & Vignais, P. V. (1991) Biochem. Biophys. Res. Commun. 180, 615-622]. Optimal conditions for the ADP-ribosylation of the C3 substrate have been established in order to follow the course of its purification from bovine neutrophil cytosol. In particular, phosphoinositides at micromolar concentrations were found to enhance the ADP-ribosylation capacity of the C3 substrate in crude neutrophil cytosol and partially purified fractions. A [32P]ADP-ribosylatable protein, migrating on SDS-PAGE with a mass of 24 kDa, was copurified with a 29-kDa protein by a series of chromatographic steps on DEAE-Sephacel, Biogel P60, and Mono Q. In the case of the C3 substrate, isoelectric focusing revealed two major labeled bands with pI values of 6.2 and 5.6; the pI of the 29-kDa protein was 4.8-5.0. On the basis of the amino acid sequence of peptides resolved after proteolytic digestion, the 24-kDa protein and the 29-kDa protein were identified respectively as rho and the GDP dissociation inhibitor (GDI), suggesting that rho and GDI copurify from bovine neutrophil cytosol in the form of a complex. The presence of a number of amino acid residues specific of rho A in the enzymatic digest originating from rho indicates that, among the rho proteins, at least rho A belongs to the GDI-rho complex.     

Isolation of human neutrophil plasma membranes employing neutrophil cytoplasts and changes in the cell-surface proteins upon cell activation

A plasma membrane fraction, highly enriched in 5'-nucleotidase activity, was prepared from human neutrophils by disruption of previously formed neutrophil cytoplasts (enucleated neutrophils), which were devoid of intracellular organelles. This plasma membrane fraction shows an extremely low contamination by specific and azurophilic granule markers as compared to previous reported preparations. Nevertheless, a novel tertiary granule (Mollinedo, F. and Schneider, D.L. (1984) J. Biol. Chem. 259, 7143-7150), unlike specific and azurophilic granules, fuses partially with the cell surface under the experimental conditions used for cytoplast preparation. Comparison between the external cell-surface proteins in resting neutrophils and neutrophil cytoplasts by lactoperoxidase-catalyzed iodination showed some differences both in deletion and in addition of proteins. In resting cells, iodine was incorporated into at least 13 proteins ranging in size from over 200 to 30 kDa. A 140 kDa polypeptide, representing the major labeled surface component in resting neutrophils, was absent from cytoplasts. Furthermore, high-molecular-weight proteins (110 and over 160 kDa were more exposed to iodination after cytoplast preparation. Activation of human neutrophils by N-formylmethionylleucylphenylalanine induced some alterations in the pattern of labeled cell-surface proteins, which correlated to a certain degree with those observed during cytoplast preparation.     

Priming-induced localization of G(ialpha2) in high density membrane microdomains

Subcellular fractionation of human neutrophils on linear sucrose density gradients was utilized to test the hypothesis that priming regulates the subcellular and sub-plasma membrane distribution of neutrophil G-protein subunits, G(ialpha2) and G(ialpha3), N-formyl peptide receptor, Lyn kinase and phospholipase C(beta2). G(ialpha2), but not G(ialpha3), moved from a lighter to a higher density plasma membrane fraction. Unoccupied N-formyl peptide receptors were found throughout the plasma membrane fractions and this distribution did not change with priming. In unprimed cells G(ialpha2) and its effector, phospholipase C(beta2), were segregated in different membrane compartments; priming caused G(ialpha2) to move to the compartment in which phospholipase C(beta2) resided. Thus, an important component of the mechanism of priming may involve regulation of the location of G-proteins and effector molecules in plasma membrane compartments where their abilities to couple may be enhanced.     

Binding and metabolism of leukotriene B4 by neutrophils and their subcellular organelles

The subcellular distribution of leukotriene (LT)B4 binding and metabolizing sites was investigated in human neutrophils. Cells were disrupted by nitrogen cavitation and fractionated by Percoll density gradient centrifugation to yield cytoplasm, membranes, azurophilic granules, and specific granules. Only membrane fractions contained high affinity [3H]LTB4 binding sites. Binding of radiolabeled ligand to membranes was rapid, reversible, and saturable; it was blocked by a series of LTB4 analogues at concentrations corresponding to their respective potencies in 1) blocking [3H]LTB4 binding to whole cells and 2) stimulating neutrophil degranulation responses. In contrast, [3H]LTB4 was metabolized by fractions enriched with markers for cytoplasm plus endoplasmic reticulum. The metabolic activity was sedimented by ultracentrifugation, enhanced by NADPH, and inhibited at 4 degrees C. The cell-free system, like intact cells, metabolized [3H]LTB4 to omega-oxidized product rapidly and quantitatively at 37 degrees C but was inactive at 4 degrees C. Whole cells converted radiolabel to 20-hydroxy (approximately 30% of product) and 20-carboxy (approximately 70% of product) derivatives; the cell-free system formed principally 20-hydroxy-[3H]LTB4. These products were less bioactive than LTB4. Nevertheless, metabolism of LTB4 played little role in limiting the cells' response to the ligand: neutrophils completed degranulation and became desensitized to LTB4 within 3-5 min of exposure. Within this time frame, they oxidized less than 30% of the stimulus, and the extracellular fluid of these neutrophil suspensions was fully capable of activating fresh cells. We conclude that neutrophils transmit bioactions of LTB4 via a specific receptor integrally associated with their plasmalemma and/or endoplasmic reticulum. They inactivate the stimulus via a particulate omega-oxidase. At the level of the individual cell, receptor down-regulation, rather than ligand metabolism, appears to limit functional responses such as degranulation.     

The type II insulin-like growth factor receptor is internalized and recycles in the absence of ligand

Recent studies have demonstrated that ligand-bound insulin-like growth factor (IGF)-II receptors on the adipocyte cell surface are rapidly internalized into an intracellular membrane fraction prior to recycling to the plasma membrane (Oka, Y., Rozek, L. M., and Czech, M. P. (1985) J. Biol. Chem. 260, 9435-9442). In order to evaluate whether these subcellular movements of IGF-II receptors in fat cells require their binding to ligand, cell surface IGF-II receptors of insulin-treated fat cells were iodinated with Na125I and lactoperoxidase at 15 degrees C. IGF-II receptors were then localized by immunoadsorption from solubilized cell surface plasma membranes and intracellular low density microsomes derived from labeled cells. When fat cells were homogenized immediately after iodination, most of the labeled IGF-II receptors were associated with the plasma membrane fraction. However, when iodinated fat cells were incubated at 37 degrees C for various times before homogenization, labeled IGF-II receptors progressively decreased in the plasma membrane fraction and concomitantly increased in the low density microsome fraction with a half-time of about 5 min. The rate of increase of radiolabeled IGF-II receptors appearing in the low density microsomes of labeled fat cells incubated with insulin was not changed by the addition of a saturating concentration of IGF-II. These results indicate that cell surface IGF-II receptors are rapidly internalized and recycled even in the absence of ligand binding in insulin-treated adipocytes.     

The fate of the N-formyl-chemotactic peptide receptor in stimulated human granulocytes: subcellular fractionation studies

Experiments were performed to examine how human granulocytes, stimulated by N-formyl-chemotactic peptides, process the N-formyl peptide receptor. One percent of the surface N-formyl-chemotactic peptide receptors of purified human granulocytes were covalently, specifically, and radioactively labeled at 4 degrees C using the photochemically reactive N-formyl-chemotactic hexapeptide CHO-Nle-Leu-Phe-Nle-[125I] Tyr-N epsilon (6-(4'-azido-2'-nitrophenyl-amino)hexanoyl)-Lys. After incubation in the presence of 500 nM of N-formyl-Met-Leu-Phe at 37 degrees C, the cells were lysed and fractionated by isopycnic surcrose density gradient sedimentation. Receptor-associated radioactivity cosedimented with plasma membrane in fractions from cells kept at 4 degrees C or incubated at 37 degrees C for 2 min or less. Fractionation of cells incubated at 37 degrees C for longer times revealed that the radioactivity sedimented to lower densities coincident with Golgi markers and the site of noncovalently bound and internalized formyl-chemotactic peptide. To follow the redistribution of unoccupied receptors, human granulocytes were stimulated with 500 nM N-formyl-Met-Leu-Phe at 37 degrees C for 5 min, washed, lysed by N2 cavitation, and fractionated by rate zonal sucrose density gradient sedimentation. Compared to unstimulated controls the specific binding of N-formyl-Met-Leu-[3H]Phe decreased 76% +/- 9% in plasma membrane fractions. N-formyl-Met-Leu-[3H]Phe-binding activity associated with an intracellular pool cosedimenting with specific granules remained unchanged. Approximately 20% of the activity lost in the plasma membrane could be accounted for by a redistribution of specific N-formyl-Met-Leu-Phe binding to fractions enriched in azurophil granules. We conclude that the receptor is the carrier in the internalization of the N-formyl-chemotactic peptides to a Golgi-enriched fraction and hypothesize that after a short residency in this fraction, the receptor may dissociate from the ligand and pass onto a fraction cosedimenting with dense granules.     

Reduction of cyclic AMP phosphodiesterase activity of several subcellular fractions of rat brain after pretreatment with phospholipase C

Cyclic AMP phosphodiesterase (PDE) activity was assayed in the plasma membrane, mitochondrial and microsomal fractions of rat brain. The specific activity of the enzyme was highest in the plasma membrane fraction followed by mitochondrial and then the microsomal fraction. Phosphodiesterase activity of all three fractions was reduced after pretreatment with lecithinase C (PCase) from Clostridium perfringens but less markedly affected by the pretreatment with sphingomyelinase (SMase) from human placenta. The PDE activity of the plasma membrane fraction was more sensitive to PCase treatment compared with the other two particulate fractions, which showed only a slight loss of activity. Temperature seemed to affect PDE activity of the plasma membrane. The enzyme was quite stable at 30 degrees C but its activity dropped by approximately 46% at 37 degrees C after 90 min of incubation. Pretreatment of the plasma membrane at 30 degrees C with PCase at a concentration of more than 5 U caused a marked loss of PDE activity and the decrease in activity reached a plateau at concentrations above 10 U.     

The neutrophil glycoprotein Mo1 is an integral membrane protein of plasma membranes and specific granules

The glycoprotein Mo1 has previously been demonstrated to be on the cell surface and in the specific granule fraction of neutrophils and to be translocated to the cell surface during degranulation. It is not known, however, whether Mo1 is an integral membrane protein or a soluble, intragranular constituent loosely associated with the specific granule membrane. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions enriched for azurophilic granules, specific granules, plasma membrane, and cytosol, respectively. The glycoproteins in these fractions were labeled with 3H-borohydride reduction, extracted with Triton X-114, and immunoprecipitated with 60.3, an anti-Mo1 monoclonal antibody Mo1 was detected only in the specific granule and plasma membrane fractions and partitioned exclusively into the detergent-rich fraction consistent with Mo1 being an integral membrane protein. In addition, treatment of specific granule membranes with a high salt, high urea buffer to remove absorbed or peripheral proteins failed to dissociate Mo1. These data support the hypothesis that Mo1 is an integral membrane protein of plasma and specific granule membranes in human neutrophils.     

Localization of transferrin receptors and insulin-like growth factor II receptors in vesicles from 3T3-L1 adipocytes that contain intracellular glucose transporters

Transferrin receptors in detergent extracts of subcellular membrane fractions prepared from 3T3-L1 adipocytes were measured by a binding assay. There was a small but significant increase (1.2-fold) in the amount of receptor in a crude plasma membrane fraction and a 40% decrease in the number of transferrin receptors in microsomal membranes prepared from insulin-treated cells, when compared with corresponding fractions from control cells. Intracellular vesicles containing insulin-responsive glucose transporters (GT) have been isolated by immunoadsorption from the microsomal fraction (Biber, J. W., and G. E. Lienhard. 1986. J. Biol. Chem. 261:16180-16184). All of the transferrin receptors in this fraction were localized in these vesicles; however, because the GT vesicles contain approximately 30-fold fewer transferrin receptors than GT, on the average only one vesicle in three contains a transferrin receptor. The binding of 125I-pentamannose 6-phosphate BSA to 3T3-L1 adipocytes at 4 degrees C was used to monitor surface insulin-like growth factor II (IGF-II)/mannose 6-phosphate receptors. Exposure of cells to insulin at 37 degrees C for 5 min resulted in a 2.5-4.5-fold increase in surface receptors. There was a corresponding 20% decrease in the amount of IGF-II receptors in the microsomal membranes prepared from insulin-treated cells, as assayed by immunoblotting. Moreover, the IGF-II receptors and GT were located in the same intracellular vesicles, since antibodies to the carboxyterminal peptide of either protein immunoadsorbed vesicles containing 70-95% of both proteins initially present in the microsomal fraction. In conjunction with other studies, these results indicate that in 3T3-L1 adipocytes, three membrane proteins (the GT, the transferrin receptor, and the IGF-II receptor) respond similarly to insulin, by redistributing to the surface from intracellular compartment(s) in which they are colocalized.     

Luteinizing Hormone Receptors are Confined in Mesoscale Plasma Membrane Microdomains Throughout Recovery from Receptor Desensitization

We examined the involvement of membrane microdomains during human luteinizing hormone (LH) receptor recovery from receptor desensitization after removal of bound hormone. Lateral motions of individual desensitized LH receptors expressed on the surface of Chinese hamster ovary cells and transient association of these receptors with detergent-resistant membrane (DRM) microdomains isolated using isopycnic sucrose gradient ultracentrifugation were assessed. Single particle tracking experiments showed untreated individual LH receptors to be confined within cell-surface membrane compartments with an average diameter of 199 ± 17 nm and associated with membrane fractions characteristic of bulk plasma membrane. After brief exposure to human chorionic gonadotropin (hCG), LH receptors remained for several hours desensitized to hCG challenge. Throughout this period, significantly increased numbers of LH receptors were confined within smaller diameter (<120 nm) membrane compartments and associated with DRM fragments of characteristically low density. By 5 h, when cells again produced cAMP in response to hCG, unoccupied LH receptors were found in larger 169 ± 22 nm diameter cell-surface membrane compartments and >90 % of LH receptors were again found in high-density membrane fragments characteristic of bulk plasma membrane. Taken together, these results suggest that, during recovery from LH receptor desensitization, LH receptors are both located with DRM lipid environments and confined within small, mesoscale (80–160 nm) cell-surface compartments. This may reflect hormone-driven translocation of receptors into DRM and formation there of protein aggregates too large or too rigid to permit effective signaling. Once bound hormone is removed, receptor structures would have to dissociate before receptors can again signal effectively in response to hormone challenge. Moreover, such larger protein complexes would be more easily constrained laterally by membrane structural elements and so appear resident in smaller cell-surface compartments.     

Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils

In this report, the modulation and localization of complement receptors CR1 and CR3 in neutrophils were examined with the use of monoclonal antibodies (mab) directed against these membrane proteins. We first studied complement receptor modulation in a patient with neutrophil-specific granule deficiency. With flow cytometric analysis, we determined that, while N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) (10(-6) M) caused an increase in the binding of both anti-CR1 and anti-CR3 mab to normal neutrophils, the fmet-leu-phe-stimulated neutrophils from our patient increased anti-CR1 binding but decreased anti-CR3 binding. This suggested that CR3, but not CR1, might be associated with specific granules. We next studied receptor modulation in organelle-depleted neutrophil cytoplasts obtained from normal donors. Unlike the specific granule-deficient neutrophils, the normal cytoplasts failed to augment expression of either receptor after stimulation. Immunofluorescence studies of permeabilized polymorphonuclear leukocytes (PMN) revealed considerable internal binding of both anti-CR1 and anti-CR3. In additional studies, phorbol myristate acetate (PMA) was used as a stimulus for receptor modulation in normal neutrophils. Unlike fmet-leu-phe and C5a, PMA elicited a biphasic dose-response curve. High doses of PMA (greater than 0.5 ng/ml) caused a reduction in the magnitude of membrane expression of both CR1 and CR3. In studies designed to localize the internal pool of receptors, we evaluated the binding of 125I-anti-receptor mab to plasma membrane-, specific granule, and azurophilic granule-enriched fractions obtained from sucrose gradient fractionation of disrupted neutrophils. 125I-anti-CR1 mab bound to the membrane-enriched fraction but bound little to either granule-enriched fraction. In contrast, 125I-anti-CR3 mab bound more to the specific granule-enriched fraction than to the plasma membrane-enriched fraction. Azurophilic granules showed no increased anti-CR3 binding. Immunoprecipitation of radiolabeled solubilized subcellular fractions with anti-receptor mab confirmed these findings. CR3 was present in the plasma membrane-, and specific granule-enriched fraction but not in the azurophilic granule-enriched fraction. CR1, however, was present only in the plasma membrane-enriched fraction. These data indicate that there are intracellular pools for both the CR1 and CR3, but the intracellular locations for these pools are distinct. The pool for CR3 co-sediments with specific granules, while the pool for CR1 does not. Nonetheless, a variety of stimulatory agents increase and decrease the membrane expression of both receptors in parallel.     

Insulin receptor kinase following internalization in isolated rat adipocytes

We have studied how insulin-mediated internalization of insulin receptors and insulin activation of the insulin receptor kinase might be inter-related. Isolated rat adipocytes were exposed to 0, 6, or 500 ng/ml insulin for 40 min at 37 degrees C. Subsequently, plasma membrane, low-density microsomal membrane and high-density microsomal membrane subcellular fractions were prepared. Measurement of insulin binding to insulin receptors isolated from the membrane fractions revealed that exposure of cells to insulin resulted in a loss of binding activity (13% at 6 ng/ml, 27% at 500 ng/ml insulin) from the plasma membranes which was completely accounted for by the appearance of receptors in the low-density and high-density microsomal membrane fractions, indicating that insulin had induced translocation of insulin receptors from the surface to the cell interior. Measurement of kinase activity of the isolated receptors revealed that exposure of intact cells to 500 ng/ml insulin resulted in as much as a 35-fold increase in the intrinsic kinase activity of receptors from subcellular fractions. The kinase activity per receptor was equal in all fractions at 3-4 min but by 20 min the activity of the internalized receptors fell approximately 40% to a steady state; plasma membrane receptors, on the other hand, remained fully active over time. This indicates that newly internalized receptors retain their kinase activity but undergo subsequent deactivation. Following exposure of cells to 6 ng/ml insulin, the degree of activation of the insulin receptor kinase was lower in the plasma membrane fraction (24% of the insulin effect at 500 ng/ml) than in the low-density and high-density microsomal membrane fractions (54 and 77%, respectively, of the insulin effect at 500 ng/ml). These results suggest that receptors with an activated kinase are preferentially internalized. We conclude that exposure of adipocytes to insulin causes endocytosis of insulin receptors and activation of insulin receptor kinase, newly internalized receptors are fully active tyrosine kinases but are deactivated as they traverse the intracellular organelles represented by low-density and high-density microsomal membranes, and insulin receptor occupancy, possibly by stimulating phosphorylation and activating the insulin receptor kinase, is important for targeting insulin receptors for internalization.     

Metabolism of photoaffinity-labeled insulin receptors by adipocytes. Role of internalization, degradation, and recycling

Insulin receptors on isolated rat adipocytes were photoaffinity-labeled with a biologically active photo-derivative of insulin (iodinated B2 (2-nitro-4-azidophenylacetyl)-des- PheB1 -insulin) in order to study the metabolism of surface receptors after binding insulin. Adipocytes were incubated with iodinated B2 (2-nitro-4-azidophenylacetyl)-des- PheB1 -insulin (40 ng/ml) at 16 degrees C until specific binding reached equilibrium, subjected to photolysis, and then incubated at 37 degrees C to follow the metabolism of the covalent insulin-receptor complexes. Susceptibility of labeled insulin receptors to tryptic digestion was used to distinguish between receptors on the cell surface and those inside the cell. Following incubation of photoaffinity-labeled adipocytes at 37 degrees C, there was an initial rapid loss of insulin receptors from the cell surface. The internalization of insulin receptors occurred at a significantly faster rate than the loss of receptors from the cell, resulting in an accumulation of intracellular receptors. The proportion of surface-derived receptors inside the cell reached an apparent steady state after 30 min and represented about 20% of the labeled receptors originally on the cell surface. Chloroquine had no effect on the internalization of insulin receptors but inhibited their degradation. Cycloheximide inhibited both internalization and degradation of insulin receptors. After 60 min at 37 degrees C, the disappearance of insulin receptors from the cell surface slowed markedly and the overall loss of insulin receptors from the cell was minimal. If chloroquine was added at this time, there was a marked increase in the loss of receptors from the cell surface with a concomitant 2-fold increase in the intracellular pool of surface-derived receptors. From these observations, we conclude that 1) internalization is not rate-limiting in insulin receptor degradation, 2) chloroquine has no effect on the internalization of insulin receptors but inhibits the intracellular degradation of receptors, 3) cycloheximide interferes with both the internalization and degradation of insulin receptors, and 4) the plateau in the loss of labeled receptors from the cell surface after 60 min at 37 degrees C could be due to a new steady state balance between internalization and recycling of photoaffinity-labeled receptors.     

Internalization of insulin and its receptor in the isolated rat adipose cell. Time-course and insulin concentration dependency

The time-course and insulin concentration dependency of internalization of insulin and its receptor have been examined in isolated rat adipose cells at 37 degrees C. The internalization of insulin was assessed by examining the subcellular distribution of cell-associated [125I]insulin among plasma membrane, and high-density (endoplasmic reticulum-enriched) and low-density (Golgi-enriched) microsomal membrane fractions prepared by differential ultracentrifugation. The distribution of receptors was measured by the steady-state exchange binding of fresh [125I]insulin to these same membrane fractions. At 37 degrees C, insulin binding to intact cells is accompanied initially by the rapid appearance of intact insulin in the plasma membrane fraction, and subsequently, by its rapid appearance in both the high-density and low-density microsomal membrane fractions. An apparent steady-state distribution of insulin per mg of membrane protein among these subcellular fractions is achieved within 30 min in a ratio of 1:1.54:0.80, respectively. Concomitantly, insulin binding to intact cells is associated with the rapid disappearance of approx. 30% of the insulin receptors initially present in the plasma membrane fraction and appearance of 20-30% of those lost in the low-density microsomal membrane fraction. However, the number of receptors in the high-density microsomal membrane fraction does not change. This redistribution of receptors also appears to reach a steady-state within 30 min. Both processes are insulin concentration-dependent, correlating with receptor occupancy in the intact cell, and are partially inhibited at 16 degrees C. While the steady-state subcellular distributions of insulin and its receptor do not correlate with that of acid phosphatase, chloroquine markedly increases the levels of insulin associated with all three membrane fractions in apparent proportion to the distribution of this lysosomal marker enzyme activity, without more than marginally potentiating insulin's effects on the distribution of receptors. These results demonstrate that insulin, initially bound to the plasma membrane of the isolated rat adipose cell, is rapidly translocated by a receptor-mediated process into at least two intracellular compartments associated with the cell's high- and low-density microsomes. Furthermore, insulin simultaneously induces the translocation of its own receptor from the plasma membrane into the latter compartment. These translocations appear to represent the internalization and partial dissociation of the insulin-receptor complex through insulin-induced receptor cycling.