Protein sorting among two distinct export pathways occurs from the content of maturing exocrine storage granules.

We have developed a method for separating purified parotid secretory granules according to their degree of maturation, and we have used this method to examine the relationship between granule formation and stimulus-independent (constitutive) protein secretion. Constitutive export of pulse-labeled secretory proteins occurs almost entirely after their appearance in newly formed granules, and this secretion can be resolved kinetically into two distinct components. Later-phase secretion is the more prominent component and, according to kinetic and compositional criteria, appears to result from basal exocytosis of mature granules. In contrast, early-phase secretion (1.5-15% of constitutive protein output) appears to originate from maturing granules but differs significantly from granule content in composition; that is, the early component exports individual protein species in different relative amounts. Maturing granules, which are labeled most highly before and during the appearance of early-phase secretion, possess numerous coated membrane evaginations suggestive of vesicular traffic. We propose that, in addition to basal exocytosis of relatively mature granules, constitutive exocrine secretion results from limited, selective removal of content proteins from forming and maturing granules. Thus protein sorting and packaging occur together in granule compartments. Exocrine secretory granules constitute an extension of the post-Golgi sorting system and are not merely terminal depots for proximally targeted polypeptides.

Distinct dynamin-dependent and -independent mechanisms target structurally homologous dopamine receptors to different endocytic membranes.

D1 and D2 dopamine receptors are structurally homologous G protein-coupled receptors that serve distinct physiological functions both in neurons and nonneural cell types. We have observed that these receptors are selectively endocytosed in HEK293 cells by distinct dynamin-dependent and -independent mechanisms. Although these endocytic mechanisms operate with similarly rapid kinetics, they differ in their regulation by agonist and deliver D1 and D2 receptors specifically to different primary endocytic vesicles. After this segregation into different endocytic membranes, both D1 and D2 receptors recycle to the plasma membrane. Similar results are observed in Neuro2A neuroblastoma cells coexpressing both receptors at high levels. These findings establish that "classical" dynamin-dependent and "alternative" dynamin-independent endocytic mechanisms differ in their physiological regulation, sort structurally homologous signaling receptors in the plasma membrane, and mediate distinct early endocytic pathways leading to recycling endosomes. Our results also refute the previous hypothesis that dynamin-independent endocytosis targets G protein-coupled receptors selectively to lysosomes, and they suggest a new role of endocytic sorting mechanisms in physically segregating structurally homologous signaling receptors at the cell surface.

Signal transduction. A new thread in an intricate web.

Understanding how biochemical pathways are connected in the cell is one of the big challenges facing cell biologists. In a Perspective, von Zastrow and Mostov describe new work that identifies a protein called RGS-PX1 as the linchpin that connects signal transduction activated by G protein-coupled receptors with membrane trafficking events.

Functional dissociation of mu opioid receptor signaling and endocytosis: implications for the biology of opiate tolerance and addiction.

Opiate analgesia, tolerance, and addiction are mediated by drug-induced activation of the mu opioid receptor. A fundamental question in addiction biology is why exogenous opiate drugs have a high liability for inducing tolerance and addiction while native ligands do not. Studies indicate that highly addictive opiate drugs such as morphine are deficient in their ability to induce the desensitization and endocytosis of receptors. Here, we demonstrate that this regulatory mechanism reveals an independent functional property of opiate drugs that can be distinguished from previously established agonist properties. Moreover, this property correlates with agonist propensity to promote physiological tolerance, suggesting a fundamental revision of our understanding of the role of receptor endocytosis in the biology of opiate drug action and addiction.

Role of AMPA receptor cycling in synaptic transmission and plasticity.

Compounds known to disrupt exocytosis or endocytosis were introduced into CA1 pyramidal cells while monitoring excitatory postsynaptic currents (EPSCs). Disrupting exocytosis or the interaction of GluR2 with NSF caused a gradual reduction in the AMPAR EPSC, while inhibition of endocytosis caused a gradual increase in the AMPAR EPSC. These manipulations had no effect on the NMDAR EPSC but prevented the subsequent induction of LTD. These results suggest that AMPARs, but not NMDARs, cycle into and out of the synaptic membrane at a rapid rate and that certain forms of synaptic plasticity may utilize this dynamic process.

Regulation of AMPA receptor endocytosis by a signaling mechanism shared with LTD.

The endocytosis of AMPA receptors is thought to be important in the expression of long-term depression (LTD) triggered by NMDA receptor activation. Although signaling pathways necessary for LTD induction have been identified, those responsible for the regulated internalization of AMPA receptors are unknown. Here we show that activation of NMDA receptors alone can trigger AMPA receptor endocytosis through calcium influx and activation of the calcium-dependent protein phosphatase calcineurin. A distinct signaling mechanism mediates the AMPA receptor endocytosis stimulated by insulin. These results demonstrate that although multiple signaling pathways can induce AMPA receptor internalization, NMDA receptor activation enhances AMPA receptor endocytosis via a signaling mechanism required for the induction of LTD.

Rapid redistribution of glutamate receptors contributes to long-term depression in hippocampal cultures.

Synaptic strength can be altered by a variety of pre- or postsynaptic modifications. Here we test the hypothesis that long-term depression (LTD) involves a decrease in the number of glutamate receptors that are clustered at individual synapses in primary cultures of hippocampal neurons. Similar to a prominent form of LTD observed in hippocampal slices, LTD in hippocampal cultures required NMDA receptor activation and was accompanied by a decrease in the amplitude and frequency of miniature excitatory postsynaptic currents. Immunocytochemical analysis revealed that induction of LTD caused a concurrent decrease in the number of AMPA receptors clustered at synapses but had no effect on synaptic NMDA receptor clusters. These results suggest that a subtype-specific redistribution of synaptic glutamate receptors contributes to NMDA receptor-dependent LTD.

Activation-induced subcellular redistribution of Gs alpha.

We have examined the subcellular distribution of alpha s, the alpha subunit of the heterotrimeric G protein Gs, by using immunofluorescence microscopy. In transiently transfected HEK293 cells, wild-type alpha s localizes to the plasma membrane. However, a mutationally activated alpha s (alpha sR201C) is diffusely distributed throughout the cytoplasm. Similarly, cholera toxin activation of alpha s causes it to redistribute from the plasma membrane to cytoplasm in stably transfected cells. In HEK293 cells stably transfected with alpha s and the beta 2-adrenergic receptor (beta-AR), stimulation of the beta-AR by the agonist isoproterenol also causes a translocation of alpha s from the plasma membrane to cytoplasm. Replacing the agonist with antagonist allows alpha s to return to the plasma membrane, demonstrating the reversibility of alpha s translocation. Receptor-activated alpha s does not colocalize with internalized beta-AR at endosomes. Incubation of cells in hypertonic sucrose to inhibit clathrin-coated pit-mediated endocytosis of agonist-activated beta-AR failed to block agonist-stimulated redistribution of alpha s. These findings demonstrate that activated alpha s reversibly undergoes a translocation from the plasma membrane to cytoplasm and begin to address the relationship between regulated trafficking of a seven-transmembrane receptor and its cognate G protein.

Downregulation of G protein-coupled receptors.

Major advances have been made in understanding mechanisms mediating downregulation of G protein-coupled receptors. Recent studies emphasize the role of multiple proteolytic mechanisms in downregulation. A specific mechanism of downregulation, mediated by endocytosis of receptors via clathrin-coated pits followed by sorting to lysosomes, has been examined in detail. Specific protein interactions that control the specificity of G-protein-coupled receptor trafficking in this pathway are beginning to be elucidated.

ß-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation.

ß-Arrestins critically regulate G-protein-coupled receptor (GPCR) signalling, not only 'arresting' the G protein signal but also modulating endocytosis and initiating a discrete G-protein-independent signal through MAP kinase. Despite enormous recent progress towards understanding biophysical aspects of arrestin function, arrestin cell biology remains relatively poorly understood. Two key tenets underlie the prevailing current view: ß-arrestin accumulates in clathrin-coated structures (CCSs) exclusively in physical complex with its activating GPCR, and MAP kinase activation requires endocytosis of formed GPCR-ß-arrestin complexes. We show here, using ß1-adrenergic receptors, that ß-arrestin-2 (arrestin 3) accumulates robustly in CCSs after dissociating from its activating GPCR and transduces the MAP kinase signal from CCSs. Moreover, inhibiting subsequent endocytosis of CCSs enhances the clathrin- and ß-arrestin-dependent MAP kinase signal. These results demonstrate ß-arrestin 'activation at a distance', after dissociating from its activating GPCR, and signalling from CCSs. We propose a ß-arrestin signalling cycle that is catalytically activated by the GPCR and energetically coupled to the endocytic machinery.

Resonance Raman spectroscopy of chemically modified and isotopically labelled purple membranes. I. A critical examination of the carbon-nitrogen vibrational modes.

Resonance Raman spectra of bacteriorhodopsin are compared to the spectra of this protein modified in the following ways: (1) selective deuteration at the C-15 carbon atom of retinal, (2) full deuteration of the retinal, (3) the addition of a conjugated double bond in the beta-ionone ring (3-dehydroretinal), (4) full deuteration of the protein and lipid components, (5) 15N enrichment of the entire membrane and (6) deuteration of the entire membrane (including the retinal). A detailed comparison of the 15N-enriched membrane and naturally occurring purple membrane from 800 cm-1 to 1700 cm-1 reveals that 15N enrichment affects the frequency of only two vibrational modes. These occur at 1642 cm-1 and 1620 cm-1 in naturally occurring purple membrane and at 1628 cm-1 and 1615 cm-1 in the 15N-enriched samples. Therefore, this pair of bands reflects the states of protonation of the Schiff base. However, our data also indicate that neither of these modes are simple, localized C=N-H or C=N stretching vibrations. In the case of the 1642 cm-1 band motions of the retinal chain beyond C-15 are not significantly involved. On the other hand, in the 1620 cm-1 band atomic motions in the isoprenoid chain beyond C-15 are involved.

Role of endocytosis in mediating downregulation of G-protein-coupled receptors.

Many G-protein-coupled receptors (GPCRs) undergo agonist-induced endocytosis. Such endocytosis has been implicated in diverse processes of receptor regulation, including reversible sequestration of receptors in endosomes and proteolytic downregulation of receptors in lysosomes. The precise relationships between membrane pathways that mediate receptor sequestration and downregulation remain controversial. Recent studies suggest that GPCRs can be segregated within distinct microdomains of the plasma membrane before endocytosis occurs, and others suggest that certain GPCRs are sorted between divergent membrane pathways after endocytosis by clathrin-coated pits. Furthermore, emerging data implicate a specific role of the actin cytoskeleton and receptor phosphorylation in controlling endocytic sorting of a particular GPCR. In this article, recent research into endocytosis of GPCRs will be discussed together with some important and unresolved questions regarding the diversity and specificity of mechanisms that mediate downregulation of GPCRs.

Diversity and specificity in the regulated endocytic membrane trafficking of G-protein-coupled receptors.

Many G-protein-coupled receptors (GPCRs) are regulated by endocytosis. Pharmacological studies defining distinct processes of ligand-induced sequestration and down-regulation provided early evidence for significant complexity in the endocytic membrane trafficking of GPCRs. In this review, we discuss our present understanding of the diversity and specificity of GPCR endocytic membrane trafficking, focusing primarily on proteolytic down-regulation of GPCRs via delivery to lysosomes. We discuss evidence suggesting that certain GPCRs can be targeted selectively to lysosomes after endocytosis by the same membrane pathway that mediates the process of rapid sequestration, and we highlight recent progress in understanding a mechanism that controls the sorting of a specific GPCR between distinct membrane pathways after endocytosis by clathrin-coated pits.

Tales from the crypt: evidence for heptahelical receptor signaling in the endocytic pathway.

In certain circumstances, internalized receptors are able to continue signaling after endocytosis. Whistler et al. discuss how the interaction of heterotrimeric GTP-binding protein (G protein)-coupled receptors with arrestins and their subsequent endocytosis may contribute to activation of the mitogen-activated protein kinase pathway. This perspective delves into the role that scaffolds may play in organizing and specifying downstream signaling events that occur after internalization of G protein-coupled receptors.

Similarities and differences among neuroendocrine, exocrine, and endocytic vesicles.

Secretory and endocytic vesicles have analogous functions as cyclic carriers between specific cellular compartments. The centrifugally functioning secretory system operates from the Golgi complex, whereas the centripetally functioning endocytic system operates from the cell surface. Further, within polarized epithelial cells the export traffic can be directed to a distinct plasmalemmal domain which distinguishes exocrine from endocrine secretion and import traffic can be directed transcellularly. These shuttle operations involve a special class of lipid-rich, protein-poor membranes that appear to use an inwardly directed H+-translocase activity to varying extents for pH-dependent sorting and for accumulation and concentration of transported molecules. Comparative analyses of purified membrane preparations from exocrine and endocrine sources identify compositional overlap between different types of shuttle membrane. However, the structural elements that specify a particular origin or destination for a given carrier or determine function in storage and stimulus-dependent shuttling remain unknown.

Role of endocytosis in signalling and regulation of G-protein-coupled receptors.

Many G-protein-coupled receptors (GPCRs) undergo agonist-induced endocytosis. Endocytosis contributes to distinct processes that regulate the number and functional activity of receptors present in the plasma membrane, contributing to the well described processes of receptor sequestration and down-regulation. Emerging evidence suggests additional functions of endocytosis in mediating GPCR signalling via certain effector pathways, such as mitogen-activated protein kinase modules. The diverse functions of endocytosis raise fundamental questions about the nature of the vesicular carriers and membrane pathways that mediate the endocytic trafficking of specific GPCRs. Insights into the biochemical and functional properties of endocytic vesicles containing internalized opioid and adrenergic receptors will be discussed. Progress towards understanding the mechanisms that control the specificity with which distinct GPCRs are sorted to specialized subpopulations of endocytic vesicles will be highlighted.

Endocytosis and downregulation of G protein-coupled receptors.

The number of G protein-coupled receptors (GPCRs) displayed at the cell surface is a critical determinant of physiological responsiveness to native ligands and drugs. Downregulation of the number of adrenergic and dopaminergic receptors present on specific neurons can be induced by receptor agonists or by drugs that increase extracellular concentrations of catecholamines such as dopamine. Thus agonist-induced downregulation of GPCRs is of potentially great importance to the treatment of Parkinson's Disease. However, little is known about biochemical mechanisms that mediate GPCR downregulation. Recent studies of cloned GPCRs have provided exciting insights into specific mechanisms that control endocytosis of receptors from the plasma membrane and regulate proteolytic degradation of receptors. In this review we briefly survey representative studies establishing that multiple mechanisms of GPCR membrane trafficking can function in downregulation function both in neural and non-neural cell types. Then we focus on our present view of mechanisms mediating regulated proteolysis of GPCRs, highlighting recent progress in understanding membrane trafficking of GPCRs from the cell surface to lysosomes. Finally we discuss emerging evidence regarding a specific mechanism that modulates sorting of certain GPCRs between recycling and degradative pathways.

Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors.

Agonist-regulated redistribution of human beta 2-adrenergic receptors was examined in 293 cells. A specific antiserum recognizing the carboxyl-terminal hydrophilic domain of the receptor was developed, characterized, and used for immunocytochemical localization of receptors in fixed cells by conventional fluorescence and confocal fluorescence microscopy. The beta-adrenergic agonist isoproterenol induced redistribution of receptors from the surface of cells into small (less than 1 micron diameter) punctuate accumulations which were detected in cells within 2 min of agonist addition. The time course of receptor redistribution paralleled that of receptor sequestration measured by ligand binding, and receptor redistribution was reversible in the presence of the beta-adrenergic antagonist alprenolol. Optical sections imaged through cells by confocal microscopy localized receptor accumulations within the cytoplasm. To address the question of receptor internalization further, a mutant receptor possessing an engineered antigenic epitope in the amino-terminal hydrophilic domain was constructed, transfected into cells, and localized using both a monoclonal antibody recognizing the epitope tag (receptor ectodomain) and an antiserum recognizing the carboxyl terminus (receptor endodomain). In untreated cells most receptor antigen was detected at the cell surface, as assessed by accessibility to ectodomain antibodies in unpermeabilized specimens. In isoproterenol-treated cells, however, little receptor antigen was detected at the cell surface. Punctate receptor accumulations present in isoproterenol-treated cells were labeled by antibodies only following permeabilization of cells, as expected if these receptor accumulations were intracellular. Finally, internalized beta-adrenergic receptors colocalized with transferrin receptors, which are markers of endosomal membranes. These data provide several lines of evidence establishing that beta-adrenergic receptors undergo ligand-regulated internalization, they suggest that internalized receptors may be recycled back to the cell surface, and they provide the first direct indication that these processes involve the same endosomal membrane system passaged by constitutively recycling receptors.

Type-specific sorting of G protein-coupled receptors after endocytosis.

The beta(2)-adrenergic receptor (B2AR) and delta-opioid receptor (DOR) are structurally distinct G protein-coupled receptors (GPCRs) that undergo rapid, agonist-induced internalization by clathrin-coated pits. We have observed that these receptors differ substantially in their membrane trafficking after endocytosis. B2AR expressed in stably transfected HEK293 cells exhibits negligible (<10%) down-regulation after continuous incubation of cells with agonist for 3 h, as assessed both by radioligand binding (to detect functional receptors) and immunoblotting (to detect total receptor protein). In contrast, DOR exhibits substantial (>/=50%) agonist-induced down-regulation when examined by similar means. Degradation of internalized DOR is sensitive to inhibitors of lysosomal proteolysis. Flow cytometric and surface biotinylation assays indicate that differential sorting of B2AR and DOR between distinct recycling and non-recycling pathways (respectively) can be detected within approximately 10 min after endocytosis, significantly before the onset of detectable proteolytic degradation of receptors ( approximately 60 min after endocytosis). Studies using pulsatile application of agonist suggest that after this sorting event occurs, later steps of membrane transport leading to lysosomal degradation of receptors do not require the continued presence of agonist in the culture medium. These observations establish that distinct GPCRs differ significantly in endocytic membrane trafficking after internalization by the same membrane mechanism, and they suggest a mechanism by which brief application of agonist can induce substantial down-regulation of receptors.