Combinatorial expression of GPCR isoforms affects signalling and drug responses.

G-protein-coupled receptors (GPCRs) are membrane proteins that modulate physiology across human tissues in response to extracellular signals. GPCR-mediated signalling can differ because of changes in the sequence1,2 or expression3 of the receptors, leading to signalling bias when comparing diverse physiological systems4. An underexplored source of such bias is the generation of functionally diverse GPCR isoforms with different patterns of expression across different tissues. Here we integrate data from human tissue-level transcriptomes, GPCR sequences and structures, proteomics, single-cell transcriptomics, population-wide genetic association studies and pharmacological experiments. We show how a single GPCR gene can diversify into several isoforms with distinct signalling properties, and how unique isoform combinations expressed in different tissues can generate distinct signalling states. Depending on their structural changes and expression patterns, some of the detected isoforms may influence cellular responses to drugs and represent new targets for developing drugs with improved tissue selectivity. Our findings highlight the need to move from a canonical to a context-specific view of GPCR signalling that considers how combinatorial expression of isoforms in a particular cell type, tissue or organism collectively influences receptor signalling and drug responses.

Location-biased activation of the proton-sensor GPR65 is uncoupled from receptor trafficking.

The canonical view of G protein-coupled receptor (GPCR) function is that receptor trafficking is tightly coupled to signaling. GPCRs remain on the plasma membrane (PM) at the cell surface until they are activated, after which they are desensitized and internalized into endosomal compartments. This canonical view presents an interesting context for proton-sensing GPCRs because they are more likely to be activated in acidic endosomal compartments than at the PM. Here, we show that the trafficking of the prototypical proton-sensor GPR65 is fully uncoupled from signaling, unlike that of other known mammalian GPCRs. GPR65 internalizes and localizes to early and late endosomes, from where they signal at steady state, irrespective of extracellular pH. Acidic extracellular environments stimulate receptor signaling at the PM in a dose-dependent manner, although endosomal GPR65 is still required for a full signaling response. Receptor mutants that were incapable of activating cAMP trafficked normally, internalize and localize to endosomal compartments. Our results show that GPR65 is constitutively active in endosomes, and suggest a model where changes in extracellular pH reprograms the spatial pattern of receptor signaling and biases the location of signaling to the cell surface.

Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains.

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.

The beta 2 adrenergic receptor cross-linked interactome identifies 14-3-3 proteins as regulating the availability of signaling-competent receptors.

The emerging picture of G protein-coupled receptor function suggests that the global signaling response is an integrated sum of a multitude of individual receptor responses, each regulated by their local protein environment. The beta 2 adrenergic receptor (B2AR) has long served as an example receptor in the development of this model. But the mechanism and the identity of the protein-protein interactions that govern the availability of receptors competent for signaling remains incompletely characterized. To address this question, we characterized the interactome of agonist-stimulated B2AR in HEK293 cells using FLAG co-immunoprecipitation coupled to SILAC labeling and mass spectrometry. Our B2AR cross-linked interactome identified 190 high-confidence proteins, including almost all known interacting proteins and six out of seven isoforms of the 14-3-3 family of scaffolding proteins. Inhibiting 14-3-3 proteins with the peptide difopein enhanced isoproterenol-stimulated adrenergic signaling via cAMP approximately three-fold, and increased both miniGs and arrestin recruitment to B2AR more than two fold each, without noticeably changing EC50 with respect to cAMP signaling or effector recruitment upon stimulation. Our results show that 14-3-3 proteins negatively regulate downstream signaling by inhibiting access of B2AR to effector proteins. We propose that 14-3-3 proteins maintain a dynamic pool of B2AR that has reduced signaling efficacy in response to acute agonist stimulation, limiting the amount of signaling-competent receptors at the plasma membrane. Significance Statement This study presents a new interactome of the agonist-stimulated beta 2 adrenergic receptor (B2AR), a paradigmatic GPCR that is both a model system for members of this class and an important signaling protein in respiratory, cardiovascular, and metabolic regulation. We identify 14-3-3 proteins as responsible for restricting B2AR access to signaling effectors and maintaining a receptor population that is insensitive to acute stimulation by agonists.

The Synthetic Cannabinoid WIN55,212-2 Can Disrupt the Golgi Apparatus Independent of Cannabinoid Receptor-1.

The synthetic cannabinoid WIN55,212-2 (WIN) is widely used as a pharmacological tool to study the biologic activity of cannabinoid receptors. In contrast to many other cannabinoid agonists, however, WIN also causes broad effects outside of neurons, such as reducing inflammatory responses, causing cell cycle arrest, and reducing general protein expression. How exactly WIN causes these broad effects is not known. Here we show that WIN partially disrupts the Golgi apparatus at nanomolar concentrations and fully disperses the Golgi apparatus in neuronal and non-neuronal cells at micromolar concentrations. WIN55,212-3, the enantiomer of WIN; JWH-018, a related alkylindole; or 2-arachidonoylglycerol, an endocannabinoid, did not cause Golgi disruption, suggesting that the effect was specific to the chirality of WIN. WIN treatment also perturbed the microtubule network. Importantly, WIN disrupted the Golgi in primary cortical neurons derived from mice where cannabinoid receptor-1 (CB1) was genetically knocked out, indicating that the effects were independent of CB1 signaling. The Golgi dispersion could not be explained by WIN's action on peroxisome proliferator-activated receptors. Our results show that WIN can disrupt the Golgi apparatus independent of CB1 in cultured cells. These effects could contribute to the unique physiologic effects that WIN exhibits in neuronal behavior, as well as its role as an antiproliferative and anti-inflammatory agent. SIGNIFICANCE STATEMENT: The synthetic cannabinoid WIN55,212-2 (WIN), widely used to investigate the cannabinoid system, also shows unique broader effects at cellular and organismal levels compared to endogenous cannabinoids. Our study shows that WIN can disrupt the Golgi apparatus and the microtubule network in multiple cell types, independent of cannabinoid receptors. These results could explain how WIN reduces surface levels of proteins and contributes to the unique physiological effects observed with WIN.

Regulation of G protein-coupled receptor signaling by plasma membrane organization and endocytosis.

The trafficking of G protein coupled-receptors (GPCRs) is one of the most exciting areas in cell biology because of recent advances demonstrating that GPCR signaling is spatially encoded. GPCRs, acting in a diverse array of physiological systems, can have differential signaling consequences depending on their subcellular localization. At the plasma membrane, GPCR organization could fine-tune the initial stages of receptor signaling by determining the magnitude of signaling and the type of effectors to which receptors can couple. This organization is mediated by the lipid composition of the plasma membrane, receptor-receptor interactions, and receptor interactions with intracellular scaffolding proteins. GPCR organization is subsequently changed by ligand binding and the regulated endocytosis of these receptors. Activated GPCRs can modulate the dynamics of their own endocytosis through changing clathrin-coated pit dynamics, and through the scaffolding adaptor protein ß-arrestin. This endocytic regulation has signaling consequences, predominantly through modulation of the MAPK cascade. This review explores what is known about receptor sorting at the plasma membrane, protein partners that control receptor endocytosis, and the ways in which receptor sorting at the plasma membrane regulates downstream trafficking and signaling.

Compartmentalized GPCR Signaling from Intracellular Membranes.

G protein-coupled receptors (GPCRs) are integral membrane proteins that transduce a wide array of inputs including light, ions, hormones, and neurotransmitters into intracellular signaling responses which underlie complex processes ranging from vision to learning and memory. Although traditionally thought to signal primarily from the cell surface, GPCRs are increasingly being recognized as capable of signaling from intracellular membrane compartments, including endosomes, the Golgi apparatus, and nuclear membranes. Remarkably, GPCR signaling from these membranes produces functional effects that are distinct from signaling from the plasma membrane, even though often the same G protein effectors and second messengers are activated. In this review, we will discuss the emerging idea of a "spatial bias" in signaling. We will present the evidence for GPCR signaling through G protein effectors from intracellular membranes, and the ways in which this signaling differs from canonical plasma membrane signaling with important implications for physiology and pharmacology. We also highlight the potential mechanisms underlying spatial bias of GPCR signaling, including how intracellular membranes and their associated lipids and proteins affect GPCR activity and signaling.

Editorial: 50 Years of Opioid Research and the International Narcotics Research Conference.

In the past 50 years, scientists have made considerable strides toward understanding how opioids act. This special issue of Molecular Pharmacology celebrates these 50 years of opioid research and the role that the International Narcotics Research Conference has played in driving this research, by bringing together review and original research articles that present historical highlights, the current state of the art, and perspectives on the future of opioid research. SIGNIFICANCE STATEMENT: Opioids have been used for thousands of years to manage pain and cause euphoria, but their use has been highly limited due to serious side effects. Deciphering the mechanisms of how opioids mediate beneficial and adverse physiological outcomes is essential for developing better treatments for pain and for opioid addiction.

A New Paroxetine-Based GRK2 Inhibitor Reduces Internalization of the µ-Opioid Receptor.

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs during heart failure, leads to the overexpression of GRKs and maladaptive downregulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or GSK180736A scaffold. A new inhibitor, CCG258747, which is based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. CCG258747 and the closely related compound CCG258208 also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of CCG258747 and 10 other inhibitors with different GRK subfamily selectivities and with either the paroxetine or GSK180736A scaffold to block internalization of the µ-opioid receptor (MOR). CCG258747 showed the best efficacy in blocking MOR internalization among the compounds tested. Furthermore, we show that compounds based on paroxetine had much better cell permeability than those based on GSK180736A, which explains why GSK180736A-based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirming that GRK2 predominantly drives internalization of MOR in the cell lines we tested and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy. SIGNIFICANCE STATEMENT: G protein-coupled receptor kinases (GRKs) are attractive targets for developing therapeutics for heart failure. We have synthesized a new GRK2 subfamily-selective inhibitor, CCG258747, which has nanomolar potency against GRK2 and excellent selectivity over other kinases. A live-cell receptor internalization assay was used to test the ability of GRK2 inhibitors to impart efficacy on a GRK-dependent process in cells. Our data indicate that CCG258747 blocked the internalization of the µ-opioid receptor most efficaciously because it has the ability to cross cell membranes.

Homologous Regulation of Mu Opioid Receptor Recycling by G ßγ , Protein Kinase C, and Receptor Phosphorylation.

Membrane trafficking and receptor signaling are two fundamental cellular processes that interact constantly. Although how trafficking regulates signaling is well studied, how signaling pathways regulate trafficking is less well understood. Here, we use the mu opioid receptor (MOR), the primary target for opioid analgesics, to define a signaling pathway that dynamically regulates postendocytic receptor recycling. By directly visualizing individual MOR recycling events, we show that agonist increases MOR recycling. Inhibition of G ßγ, phospholipase C, or protein kinase C mimicked agonist removal, whereas activation of G ßγ increased recycling even after agonist removal. Phosphorylation of serine 363 on the C-terminal tail of MOR was required and sufficient for agonist-mediated regulation of MOR recycling. Our results identify a feedback loop that regulates MOR recycling via G ßγ , protein kinase C, and receptor phosphorylation. This could serve as a general model for how signaling regulates postendocytic trafficking of G protein-coupled receptors. SIGNIFICANCE STATEMENT: G protein-coupled receptor (GPCR) localization in the endosome is being increasingly recognized as an important and distinct component of GPCR signaling and physiology. This study identifies a G protein-dependent and protein kinase C-dependent signaling pathway that dynamically regulates the endosomal localization of the mu opioid receptor, the primary target of opioid analgesics and abused drugs. This pathway could provide a mechanism to manipulate spatial encoding of opioid signaling and physiology.

A PTEN-Regulated Checkpoint Controls Surface Delivery of δ Opioid Receptors.

The δ opioid receptor (δR) is a promising alternate target for pain management because δR agonists show decreased abuse potential compared with current opioid analgesics that target the µ opioid receptor. A critical limitation in developing δR as an analgesic target, however, is that δR agonists show relatively low efficacy in vivo, requiring the use of high doses that often cause adverse effects, such as convulsions. Here we tested whether intracellular retention of δR in sensory neurons contributes to this low δR agonist efficacy in vivo by limiting surface δR expression. Using direct visualization of δR trafficking and localization, we define a phosphatase and tensin homolog (PTEN)-regulated checkpoint that retains δR in the Golgi and decreases surface delivery in rat and mice sensory neurons. PTEN inhibition releases δR from this checkpoint and stimulates delivery of exogenous and endogenous δR to the neuronal surface both in vitro and in vivo PTEN inhibition in vivo increases the percentage of TG neurons expressing δR on the surface and allows efficient δR-mediated antihyperalgesia in mice. Together, we define a critical role for PTEN in regulating the surface delivery and bioavailability of the δR, explain the low efficacy of δR agonists in vivo, and provide evidence that active δR relocation is a viable strategy to increase δR antinociception.SIGNIFICANCE STATEMENT Opioid analgesics, such as morphine, which target the µ opioid receptor (µR), have been the mainstay of pain management, but their use is highly limited by adverse effects and their variable efficacy in chronic pain. Identifying alternate analgesic targets is therefore of great significance. Although the δ opioid receptor (δR) is an attractive option, a critical limiting factor in developing δR as a target has been the low efficacy of δR agonists. Why δR agonists show low efficacy is still under debate. This study provides mechanistic and functional data that intracellular localization of δR in neurons is a key factor that contributes to low agonist efficacy, and presents a proof of mechanism that relocating δR improves efficacy.

The Proteome of BLOC-1 Genetic Defects Identifies the Arp2/3 Actin Polymerization Complex to Function Downstream of the Schizophrenia Susceptibility Factor Dysbindin at the Synapse.

Proteome modifications downstream of monogenic or polygenic disorders have the potential to uncover novel molecular mechanisms participating in pathogenesis and/or extragenic modification of phenotypic expression. We tested this idea by determining the proteome sensitive to genetic defects in a locus encoding dysbindin, a protein required for synapse biology and implicated in schizophrenia risk. We applied quantitative mass spectrometry to identify proteins expressed in neuronal cells the abundance of which was altered after downregulation of the schizophrenia susceptibility factor dysbindin (Bloc1s8) or two other dysbindin-interacting polypeptides, which assemble into the octameric biogenesis of lysosome-related organelles complex 1 (BLOC-1). We found 491 proteins sensitive to dysbindin and BLOC-1 loss of function. Gene ontology of these 491 proteins singled out the actin cytoskeleton and the actin polymerization factor, the Arp2/3 complex, as top statistical molecular pathways contained within the BLOC-1-sensitive proteome. Subunits of the Arp2/3 complex were downregulated by BLOC-1 loss of function, thus affecting actin dynamics in early endosomes of BLOC-1-deficient cells. Furthermore, we demonstrated that Arp2/3, dysbindin, and subunits of the BLOC-1 complex biochemically and genetically interact, modulating Drosophila melanogaster synapse morphology and homeostatic synaptic plasticity. Our results indicate that ontologically prioritized proteomics identifies novel pathways that modify synaptic phenotypes associated with neurodevelopmental disorder gene defects. SIGNIFICANCE STATEMENT: The mechanisms associated with schizophrenia are mostly unknown despite the increasing number of genetic loci identified that increase disease risk. We present an experimental strategy that impartially and comprehensively interrogates the proteome of neurons to identify effects of genetic mutations in a schizophrenia risk factor, dysbindin. We find that the expression of the actin polymerization complex Arp2/3 is reduced in dysbindin-deficient cells, thus affecting actin-dependent phenotypes in two cellular compartments where dysbindin resides, endosomes and presynapses. Our studies indicate that a central cellular structure affected by schizophrenia susceptibility loci is the actin cytoskeleton, an organelle necessary for synaptic function in the presynaptic and postsynaptic compartment.

Sequence-Specific Regulation of Endocytic Lifetimes Modulates Arrestin-Mediated Signaling at the µ Opioid Receptor.

Functional selectivity at the µ opioid receptor (µR), a prototypical G-protein-coupled receptor that is a physiologically relevant target for endogenous opioid neurotransmitters and analgesics, has been a major focus for drug discovery in the recent past. Functional selectivity is a cumulative effect of the magnitudes of individual signaling pathways, e.g., the Gαi-mediated and the arrestin-mediated pathways for µR. The present work tested the hypothesis that lifetimes of agonist-induced receptor-arrestin clusters at the cell surface control the magnitude of arrestin signaling, and therefore functional selectivity, at µR. We show that endomorphin-2 (EM2), an arrestin-biased ligand for µR, lengthens surface lifetimes of receptor-arrestin clusters significantly compared with morphine. The lengthening of lifetimes required two specific leucines on the C-terminal tail of µR. Mutation of these leucines to alanines decreased the magnitude of arrestin-mediated signaling by EM2 without affecting G-protein signaling, suggesting that lengthened endocytic lifetimes were required for arrestin-biased signaling by EM2. Lengthening surface lifetimes by pharmacologically slowing endocytosis was sufficient to increase arrestin-mediated signaling by both EM2 and the clinically relevant agonist morphine. Our findings show that distinct ligands can leverage specific sequence elements on µR to regulate receptor endocytic lifetimes and the magnitude of arrestin-mediated signaling, and implicate these sequences as important determinants of functional selectivity in the opioid system.

The α-Arrestin ARRDC3 Regulates the Endosomal Residence Time and Intracellular Signaling of the ß2-Adrenergic Receptor.

Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian α-arrestin family, which is predicted to share similar tertiary structure with visual-/ß-arrestins and also contains C-terminal PPXY motifs that mediate interaction with E3 ubiquitin ligases. Recently, ARRDC3 has been proposed to play a role in regulating the trafficking of G protein-coupled receptors, although mechanistic insight into this process is lacking. Here, we focused on characterizing the role of ARRDC3 in regulating the trafficking of the ß2-adrenergic receptor (ß2AR). We find that ARRDC3 primarily localizes to EEA1-positive early endosomes and directly interacts with the ß2AR in a ligand-independent manner. Although ARRDC3 has no effect on ß2AR endocytosis or degradation, it negatively regulates ß2AR entry into SNX27-occupied endosomal tubules. This results in delayed recycling of the receptor and a concomitant increase in ß2AR-dependent endosomal signaling. Thus, ARRDC3 functions as a switch to modulate the endosomal residence time and subsequent intracellular signaling of the ß2AR.

Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling.

The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor.

Src regulates sequence-dependent beta-2 adrenergic receptor recycling via cortactin phosphorylation.

The recycling of internalized signaling receptors, which has direct functional consequences, is subject to multiple sequence and biochemical requirements. Why signaling receptors recycle via a specialized pathway, unlike many other proteins that recycle by bulk, is a fundamental unanswered question. Here, we show that these specialized pathways allow selective control of signaling receptor recycling by heterologous signaling. Using assays to visualize receptor recycling in living cells, we show that the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, is regulated by Src family kinases. The target of Src is cortactin, an essential factor for B2AR sorting into specialized recycling microdomains on the endosome. Phosphorylation of a single cortactin residue, Y466, regulates the rate of fission of B2AR recycling vesicles from these microdomains and, therefore, the rate of delivery of B2AR to the cell surface. Together, our results indicate that actin-stabilized microdomains that mediate signaling receptor recycling can serve as a functional point of convergence for crosstalk between signaling pathways.

Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch.

The postendocytic recycling of signaling receptors is subject to multiple requirements. Why this is so, considering that many other proteins can recycle without apparent requirements, is a fundamental question. Here we show that cells can leverage these requirements to switch the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, between sequence-dependent and bulk recycling pathways, based on extracellular signals. This switch is determined by protein kinase A-mediated phosphorylation of B2AR on the cytoplasmic tail. The phosphorylation state of B2AR dictates its partitioning into spatially and functionally distinct endosomal microdomains mediating bulk and sequence-dependent recycling, and also regulates the rate of B2AR recycling and resensitization. Our results demonstrate that G protein-coupled receptor recycling is not always restricted to the sequence-dependent pathway, but may be reprogrammed as needed by physiological signals. Such flexible reprogramming might provide a versatile method for rapidly modulating cellular responses to extracellular signaling.

Spatially restricted G protein-coupled receptor activity via divergent endocytic compartments.

Postendocytic sorting of G protein-coupled receptors (GPCRs) is driven by their interactions between highly diverse receptor sequence motifs with their interacting proteins, such as postsynaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), zonula occludens-1 protein (zo-1) (PDZ) domain proteins. However, whether these diverse interactions provide an underlying functional specificity, in addition to driving sorting, is unknown. Here we identify GPCRs that recycle via distinct PDZ ligand/PDZ protein pairs that exploit their recycling machinery primarily for targeted endosomal localization and signaling specificity. The luteinizing hormone receptor (LHR) and ß2-adrenergic receptor (B2AR), two GPCRs sorted to the regulated recycling pathway, underwent divergent trafficking to distinct endosomal compartments. Unlike B2AR, which traffics to early endosomes (EE), LHR internalizes to distinct pre-early endosomes (pre-EEs) for its recycling. Pre-EE localization required interactions of the LHR C-terminal tail with the PDZ protein GAIP-interacting protein C terminus, inhibiting its traffic to EEs. Rerouting the LHR to EEs, or EE-localized GPCRs to pre-EEs, spatially reprograms MAPK signaling. Furthermore, LHR-mediated activation of MAPK signaling requires internalization and is maintained upon loss of the EE compartment. We propose that combinatorial specificity between GPCR sorting sequences and interacting proteins dictates an unprecedented spatiotemporal control in GPCR signal activity.

GM130 and GRASP65-dependent lateral cisternal fusion allows uniform Golgi-enzyme distribution.

The mammalian Golgi apparatus exists as stacks of cisternae that are laterally linked to form a continuous membrane ribbon, but neither the molecular requirements for, nor the purpose of, Golgi ribbon formation are known. Here, we demonstrate that ribbon formation is mediated by specific membrane-fusion events that occur during Golgi assembly, and require the Golgi proteins GM130 and GRASP65. Furthermore, these GM130 and GRASP65-dependent lateral cisternal-fusion reactions are necessary to achieve uniform distribution of enzymes in the Golgi ribbon. The membrane continuity created by ribbon formation facilitates optimal processing conditions in the biosynthetic pathway.