Clathrin-dependent mechanisms of G protein-coupled receptor endocytosis

The heptahelical G protein-coupled receptor (GPCR) family includes approximately 900 members and is the largest family of signaling receptors encoded in the mammalian genome. G protein-coupled receptors elicit cellular responses to diverse extracellular stimuli at the plasma membrane and some internalized receptors continue to signal from intracellular compartments. In addition to rapid desensitization, receptor trafficking is critical for regulation of the temporal and spatial aspects of GPCR signaling. Indeed, GPCR internalization functions to control signal termination and propagation as well as receptor resensitization. Our knowledge of the mechanisms that regulate mammalian GPCR endocytosis is based predominantly on arrestin regulation of receptors through a clathrin- and dynamin-dependent pathway. However, multiple clathrin adaptors, which recognize distinct endocytic signals, are now known to function in clathrin-mediated endocytosis of diverse cargo. Given the vast number and diversity of GPCRs, the complexity of clathrin-mediated endocytosis and the discovery of multiple clathrin adaptors, a single universal mechanism controlling endocytosis of all mammalian GPCRs is unlikely. Indeed, several recent studies now suggest that endocytosis of different GPCRs is regulated by distinct mechanisms and clathrin adaptors. In this review, we discuss the diverse mechanisms that regulate clathrin-dependent GPCR endocytosis.     

Cell-based, high-content screen for receptor internalization, recycling and intracellular trafficking

A variety of physiologically important receptors are internalized and then recycled back to the plasma membrane by the endocytic recycling compartment. These include the transferrin receptor and many G-protein coupled receptors (GPCRs). The internalization of GPCRs is a result of agonist stimulation. A cell-based fluorescent imaging assay is described that detects and quantifies the presence of fluorescently labeled receptors and macromolecules in the recycling compartment. This High Content Screening application is conducted on the ArrayScan II System that includes fluorescent reagents, imaging instrumentation and the informatics tools necessary to screen for compounds that affect receptor internalization, recycling and GPCR activation. We demonstrate the Receptor Internalization and Trafficking application by quantifying (i) the internalization and recycling of the transferrin receptor using a fluorescently labeled ligand and (ii) the internalization of a physiologically functional model GPCR, a GFP-parathyroid hormone receptor chimera. These assays give high signal-to-noise ratios, broad dynamic ranges between stimulated and unstimulated conditions and low variability across different screening runs. Thus, the Receptor Internalization and Trafficking application, in conjunction with the ArrayScan II System, forms the basis of a robust, information-rich and automated screen for GPCR activation.     

When trafficking and signaling mix: How subcellular location shapes G protein‐coupled receptor activation of heterotrimeric G proteins

G protein‐coupled receptors (GPCRs) physically connect extracellular information with intracellular signal propagation. Membrane trafficking plays a supportive role by “bookending” signaling events: movement through the secretory pathway delivers GPCRs to the cell surface where receptors can sample the extracellular environment, while endocytosis and endolysosomal membrane trafficking provide a versatile system to titrate cellular signaling potential and maintain homeostatic control. Recent evidence suggests that, in addition to these important effects, GPCR trafficking actively shapes the cellular signaling response by altering the location and timing of specific receptor‐mediated signaling reactions. Here, we review key experimental evidence underlying this expanding view, focused on GPCR signaling mediated through activation of heterotrimeric G proteins located in the cytoplasm. We then discuss lingering and emerging questions regarding the interface between GPCR signaling and trafficking.      

Identification of a novel endocytic recycling signal in the D1 dopamine receptor

A critical event determining the functional consequences of G protein-coupled receptor (GPCR) endocytosis is the molecular sorting of internalized receptors between divergent recycling and degradative membrane pathways. The D1 dopamine receptor recycles rapidly and efficiently to the plasma membrane after agonist-induced endocytosis and is remarkably resistant to proteolytic down-regulation. Whereas the mechanism mediating agonist-induced endocytosis of D1 receptors has been investigated in some detail, little is known about how receptors are sorted after endocytosis. We have identified a sequence present in the carboxyl-terminal cytoplasmic domain of the human D1 dopamine receptor that is specifically required for the efficient recycling of endocytosed receptors back to the plasma membrane. This sequence is distinct from previously identified membrane trafficking signals and is located in a proximal portion of the carboxyl-terminal cytoplasmic domain, in contrast to previously identified GPCR recycling signals present at the distal tip. Nevertheless, fusion of this sequence to the carboxyl terminus of a chimeric mutant delta opioid neuropeptide receptor is sufficient to re-route internalized receptors from lysosomal to recycling membrane pathways, defining this sequence as a bona fide endocytic recycling signal that can function in both proximal and distal locations. These results identify a novel sorting signal controlling the endocytic trafficking itinerary of a physiologically important dopamine receptor, provide the first example of such a sorting signal functioning in a proximal portion of the carboxyl-terminal cytoplasmic domain, and suggest the existence of a diverse array of sorting signals in the GPCR superfamily that mediate subtype-specific regulation of receptors via endocytic membrane trafficking.     

Rapid internalization and recycling of the human neuropeptide Y Y(1) receptor.

Desensitization of G protein-coupled receptors (GPCRs) involves receptor phosphorylation and reduction in the number of receptors at the cell surface. The neuropeptide Y (NPY) Y(1) receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y(1) receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y(1) receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y(1) receptors. Upon agonist challenge, the EGFP signal decreased rapidly (t(1/2) = 107 +/- 3 s) followed by a slow recovery. This decrease was blocked by BIBP3226, a Y(1) receptor antagonist, or by pertussis toxin, in agreement with Y(1) receptor activation. Internalization of EGFP-hNPY Y(1) receptors to acidic endosomal compartments likely accounts for the decrease in the EGFP signal, being absent after pretreatment with monensin. Concanavalin A and hypertonic sucrose, which inhibit clathrin-mediated endocytosis, blocked the decrease in fluorescence. After agonist, intracellular EGFP signals were punctate and co-localized with transferrin-Texas Red, a marker of clathrin-associated internalization and recycling, but not with LysoTracker Red, a lysosomal pathway marker, supporting receptor trafficking to recycling endosomes rather than the late endosomal/lysosomal pathway. Pulse-chase experiments revealed no receptor degradation after internalization. The slow recovery of fluorescence was unaffected by cycloheximide or actinomycin D, indicating that de novo synthesis of receptors was not limiting. Use of a multicompartment model to fit our fluorescence data allows simultaneous determination of internalization and recycling rate constants. We propose that rapid internalization of receptors via the clathrin-coated pits recycling pathway may largely account for the rapid desensitization of NPY Y(1) receptors.     

Constitutive traffic of melanocortin-4 receptor in Neuro2A cells and immortalized hypothalamic neurons

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that binds alpha-melanocyte-stimulating hormone (alpha-MSH) and has a central role in the regulation of appetite and energy expenditure. Most GPCRs are endocytosed following binding to the agonist and receptor desensitization. Other GPCRs are internalized and recycled back to the plasma membrane constitutively, in the absence of the agonist. In unstimulated neuroblastoma cells and immortalized hypothalamic neurons, epitopetagged MC4R was localized both at the plasma membrane and in an intracellular compartment. These two pools of receptors were in dynamic equilibrium, with MC4R being rapidly internalized and exocytosed. In the absence of alpha-MSH, a fraction of cell surface MC4R localized together with transferrin receptor and to clathrin-coated pits. Constitutive MC4R internalization was impaired by expression of a dominant negative dynamin mutant. Thus, MC4R is internalized together with transferrin receptor by clathrin-dependent endocytosis. Cell exposure toalpha-MSH reduced the amount of MC4R at the plasma membrane by blocking recycling of a fraction of internalized receptor, rather than by increasing its rate of endocytosis. The data indicate that, in neuronal cells, MC4R recycles constitutively and that alpha-MSH modulates MC4R residency at the plasma membrane by acting at an intracellular sorting step.     

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.     

Adaptor Protein-2 Interaction with Arrestin Regulates GPCR Recycling and Apoptosis

G protein-coupled receptors (GPCRs) are integral to cellular function in nearly all physiologic and many pathologic processes. GPCR signaling represents an intricate balance between receptor activation, inactivation (desensitization, internalization and degradation) and resensitization (recycling and de novo synthesis). Complex formation between phosphorylated GPCRs, arrestins and an ever-increasing number of effector molecules is known to regulate cellular function. Previous studies have demonstrated that, although N -formyl peptide receptor (FPR) internalization occurs in the absence of arrestins, FPR recycling is arrestin-dependent. Furthermore, FPR stimulation in the absence of arrestins leads to receptor accumulation in perinuclear endosomes and apoptosis. In this study, we show that the interaction of GPCR-bound arrestin with adaptor protein-2 (AP-2) is a critical anti-apoptotic event. In addition, AP-2 associates with the receptor-arrestin complex in perinuclear endosomes and is required for proper post-endocytic GPCR trafficking. Finally, we observed that depletion of endogenous AP-2 results in the initiation of apoptosis upon stimulation of multiple GPCRs, including P2Y purinergic receptors and CXCR2, but not CXCR4. We propose a model in which the abnormal accumulation of internalized GPCR-arrestin complexes in recycling endosomes, resulting from defective arrestin-AP-2 interactions, leads to the specific initiation of aberrant signaling pathways and apoptosis.     

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 β‐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.      

Quantitative Measurement of GPCR Endocytosis via Pulse-Chase Covalent Labeling

G protein-coupled receptors (GPCRs) play a critical role in many physiological systems and represent one of the largest families of signal-transducing receptors. The number of GPCRs at the cell surface regulates cellular responsiveness to their cognate ligands, and the number of GPCRs, in turn, is dynamically controlled by receptor endocytosis. Recent studies have demonstrated that GPCR endocytosis, in addition to affecting receptor desensitization and resensitization, contributes to acute G protein-mediated signaling. Thus, endocytic GPCR behavior has a significant impact on various aspects of physiology. In this study, we developed a novel GPCR internalization assay to facilitate characterization of endocytic GPCR behavior. We genetically engineered chimeric GPCRs by fusing HaloTag (a catalytically inactive derivative of a bacterial hydrolase) to the N-terminal end of the receptor (HT-GPCR). HaloTag has the ability to form a stable covalent bond with synthetic HaloTag ligands that contain fluorophores or a high-affinity handle (such as biotin) and the HaloTag reactive linker. We selectively labeled HT-GPCRs at the cell surface with a HaloTag PEG ligand, and this pulse-chase covalent labeling allowed us to directly monitor the relative number of internalized GPCRs after agonist stimulation. Because the endocytic activities of GPCR ligands are not necessarily correlated with their agonistic activities, applying this novel methodology to orphan GPCRs, or even to already characterized GPCRs, will increase the likelihood of identifying currently unknown ligands that have been missed by conventional pharmacological assays.     

Novel roles for arrestins in the post-endocytic trafficking of G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest family of transmembrane signaling molecules in the human genome. As such, they interact with numerous intracellular molecules, which can act either to propagate or curtail signaling from the receptor. Their primary mode of cellular activation occurs through heterotrimeric G proteins, which in turn can activate a wide spectrum of effector molecules, including phosphodiesterases, phospholipases, adenylyl cyclases and ion channels. Active GPCRs are also the target of G protein-coupled receptor kinases, which phosphorylate the receptors culminating in the binding of the protein arrestin. This results in rapid desensitization through inhibition of G protein binding, as well as novel mechanisms of cellular activation that involve the scaffolding of cellular kinases to GPCR-arrestin complexes. Arrestins can also serve to mediate the internalization of certain GPCRs, a process which plays an important role in regulating cellular activity both by mediating long-term desensitization through down regulation (degradation) of receptors and by recycling desensitized receptors back to the cell surface to initiate additional rounds of signaling. The mechanisms that regulate the subsequent intracellular trafficking of GPCRs following internalization are largely unknown. Recently however, it has become clear that the pattern of receptor phosphorylation and subsequent binding of arrestin play a critical role in the intracellular trafficking of internalized receptors, thereby dictating the ultimate fate of the receptor. In addition, arrestins have now been shown to be required for the recycling of GPCRs that are capable of internalizing through arrestin-independent mechanisms. This review will summarize recent advances in our understanding of the roles of arrestins in post-endocytic GPCR trafficking.     

Mechanisms regulating membrane trafficking of G protein-coupled receptors in the endocytic pathway

Endocytic membrane trafficking plays multiple roles in GPCR signaling and regulation. In the past several years much has been learned about molecular mechanisms that mediate and regulate endocytic trafficking of cloned GPCRs expressed in transfected cell lines, and there is accelerating progress toward elucidating the membrane trafficking of GPCRs in native tissues. Current views regarding ligand-induced endocytosis of adrenergic catecholamine and opioid neuropeptide receptors will be reviewed, focusing on recent data suggesting the existence of additional machinery controlling the endocytosis of specific GPCRs via clathrin-coated pits. Evidence that GPCRs are selectively 'sorted' between divergent downstream pathways after endocytosis will be discussed, focusing on recent insight to mechanisms controlling receptor sorting between distinct recycling and non-recycling membrane pathways.     

Reversible translocation of p115-RhoGEF by G(12/13)-coupled receptors

G protein-coupled receptors (GPCRs) are important targets for medicinal agents. Four different G protein families, G(s), G(i), G(q), and G(12), engage in their linkage to activation of receptor-specific signal transduction pathways. G(12) proteins were more recently studied, and upon activation by GPCRs they mediate activation of RhoGTPase guanine nucleotide exchange factors (RhoGEFs), which in turn activate the small GTPase RhoA. RhoA is involved in many cellular and physiological aspects, and a dysfunction of the G(12/13)-Rho pathway can lead to hypertension, cardiovascular diseases, stroke, impaired wound healing and immune cell functions, cancer progression and metastasis, or asthma. In this study, regulator of G protein signaling (RGS) domain-containing RhoGEFs were tagged with enhanced green fluorescent protein (EGFP) to detect their subcellular localization and translocation upon receptor activation. Constitutively active Galpha(12) and Galpha(13) mutants induced redistribution of these RhoGEFs from the cytosol to the plasma membrane. Furthermore, a pronounced and rapid translocation of p115-RhoGEF from the cytosol to the plasma membrane was observed upon activation of several G(12/13)-coupled GPCRs in a cell type-independent fashion. Plasma membrane translocation of p115-RhoGEF stimulated by a GPCR agonist could be completely and rapidly reversed by subsequent application of an antagonist for the respective GPCR, that is, p115-RhoGEF relocated back to the cytosol. The translocation of RhoGEF by G(12/13)-linked GPCRs can be quantified and therefore used for pharmacological studies of the pathway, and to discover active compounds in a G(12/13)-related disease context.     

Potent Anti-HIV Chemokine Analogs Direct Post-Endocytic Sorting of CCR5

G protein-coupled receptors (GPCRs) are desensitized and internalized following activation. They are then subjected to post-endocytic sorting (degradation, slow recycling or fast recycling). The majority of research on post-endocytic sorting has focused on the role of sequence-encoded address structures on receptors. This study focuses on trafficking of CCR5, a GPCR chemokine receptor and the principal entry coreceptor for HIV. Using Chinese Hamster Ovary cells stably expressing CCR5 we show that two different anti-HIV chemokine analogs, PSC-RANTES and 5P14-RANTES, direct receptor trafficking into two distinct subcellular compartments: the trans-Golgi network and the endosome recycling compartment, respectively. Our results indicate that a likely mechanism for ligand-directed sorting of CCR5 involves capacity of the chemokine analogs to elicit the formation of durable complexes of CCR5 and arrestin2 (beta-arrestin-1), with PSC-RANTES eliciting durable association in contrast to 5P14-RANTES, which elicits only transient association.     

A Constitutively Internalizing and Recycling Mutant of the μ-Opioid Receptor

Abstract: Internalization and recycling of G protein-coupled receptors (GPCRs), such as the μ-opioid receptor, largely depend on agonist stimulation, whereas certain other receptor types recycle constitutively, e.g., the transferrin receptor. To investigate structural domains involved in μ-opioid receptor internalization, we constructed two truncation mutants bracketing a Ser/Thr-rich domain (³⁵⁴ThrSerSerThrIleGluGlnGlnAsn³⁶²) unique to the C-terminus of the μ-opioid receptor (mutants Trunc354 and Trunc363). Ligand binding did not differ substantially, and G protein coupling was slightly lower for these μ-receptor constructs, in particular for Trunc363. To permit localization of the receptor by immunocytochemistry, an epitope tag was added to the N-terminus of the wildtype and mutant receptors. Both the wild-type μ-opioid receptor and Trunc363 resided largely at the plasma membrane and internalized into vesicles upon stimulation with the agonist [d -Ala²,N-Me-Phe⁴,Gly-ol⁵]-enkephalin. Internalization occurred into vesicles that contain transferrin receptors, as shown previously, as well as clathrin, but not caveolin. In contrast, even without any agonist present, Trunc354 colocalized in intracellular vesicles with clathrin and transferrin receptors, but not caveolin. On blocking internalization by hyperosmolar sucrose or acid treatment, Trunc354 translocated to the plasma membrane, indicating that the mutant internalized into clathrin-coated vesicles and recycled constitutively. Despite agonist-independent internalization of Trunc354, basal G protein coupling was not elevated, suggesting distinct mechanisms for coupling and internalization. Furthermore, a portion of the C-terminus, particularly the Ser/Thr domain, appears to suppress μ-receptor internalization, which can be overcome by agonist stimulation. These results demonstrate that a mutant GPCR can be constructed such that internalization, normally an agonist-dependent process, can occur spontaneously without concomitant G protein activation.     

Unconventional secretion of tissue transglutaminase involves phospholipid-dependent delivery into recycling endosomes

Although endosomal compartments have been suggested to play a role in unconventional protein secretion, there is scarce experimental evidence for such involvement. Here we report that recycling endosomes are essential for externalization of cytoplasmic secretory protein tissue transglutaminase (tTG). The de novo synthesized cytoplasmic tTG does not follow the classical ER/Golgi-dependent secretion pathway, but is targeted to perinuclear recycling endosomes, and is delivered inside these vesicles prior to externalization. On its route to the cell surface tTG interacts with internalized β1 integrins inside the recycling endosomes and is secreted as a complex with recycled β1 integrins. Inactivation of recycling endosomes, blocking endosome fusion with the plasma membrane, or downregulation of Rab11 GTPase that controls outbound trafficking of perinuclear recycling endosomes, all abrogate tTG secretion. The initial recruitment of cytoplasmic tTG to recycling endosomes and subsequent externalization depend on its binding to phosphoinositides on endosomal membranes. These findings begin to unravel the unconventional mechanism of tTG secretion which utilizes the long loop of endosomal recycling pathway and indicate involvement of endosomal trafficking in non-classical protein secretion.     

Agonist-dependent immobilization of chimeric bombesin/GRP receptors: dependence on c-Src activity and dissociation from internalization.

G-protein-coupled receptors (GPCRs) are membrane proteins that exhibit a decreased mobile fraction compared to a freely mobile plasma membrane protein. Recently, interest has focused on proteins other than heterotrimeric G-proteins that interact with GPCRs as scaffolding structures that affect receptor signal transduction. In order to investigate the physical state of receptors before and after agonist, we used fluorescence recovery after photobleaching of the bombesin/gastrin-releasing peptide (GRP) receptor fused to the intrinsically fluorescent green fluorescent protein (GFP-GRP receptor) expressed in KNRK cells to measure both the fraction of mobile receptors and their diffusion rate before and after agonist stimulation. In live cells at 37 degrees C, addition of GRP (100 nM) caused a rapid decrease in GFP-GRP receptor mobile fraction from 0.8 +/- 0.1 to 0.49 +/- 0.05, which was independent of endocytosis. Concurrently, the remaining mobile GFP-GRPreceptors showed an increase in the diffusion rate with the half-time of fluorescent recovery, tau(1/2) = 46 +/- 7 s for untreated cells, decreasing to tau(1/2) = 30 +/- 6 s for cells treated with GRP. Prior treatment with the Src-specific inhibitor PP-2 (10 microM) blocked GFP-GRP receptor immobilization while treatment with the inactive analog PP-3 (10 microM) did not affect receptor immobilization. These data suggest that agonist-bound GPCR have increased plasma membrane diffusion rates but an increased affinity for immobilization into a multiprotein complex that is mediated by Src activity.     

The expanding repertoire of receptor activity modifying protein (RAMP) function

Receptor activity modifying proteins (RAMPs) associate with G-protein-coupled receptors (GPCRs) at the plasma membrane and together bind a variety of peptide ligands, serving as a communication interface between the extracellular and intracellular environments. The collection of RAMP-interacting GPCRs continues to expand and now consists of GPCRs from families A, B and C, suggesting that RAMP activity is extremely prevalent. RAMP association with GPCRs can regulate GPCR function by altering ligand binding, receptor trafficking and desensitization, and downstream signaling pathways. Here, we elaborate on these RAMP-dependent mechanisms of GPCR regulation, which provide opportunities for pharmacological intervention.     

CNIH4 Interacts with Newly Synthesized GPCR and Controls Their Export from the Endoplasmic Reticulum

The molecular mechanisms regulating G protein‐coupled receptors (GPCRs) trafficking from their site of synthesis in the endoplasmic reticulum (ER) to their site of function (the cell surface) remain poorly characterized. Using a bioluminescence resonance energy transfer‐based proteomic screen, we identified a novel GPCR‐interacting protein; the human cornichon homologue 4 (CNIH4). This previously uncharacterized protein is localized in the early secretory pathway where it interacts with members of the 3 family of GPCRs. Both overexpression and knockdown expression of CNIH4 caused the intracellular retention of GPCRs, indicating that this ER‐resident protein plays an important role in GPCR export. Overexpression of CNIH4 at low levels rescued the maturation and cell surface expression of an intracellularly retained mutant form of the β2‐adrenergic receptor, further demonstrating a positive role of CNIH4 in GPCR trafficking. Taken with the co‐immunoprecipitation of CNIH4 with Sec23 and Sec24, components of the COPII coat complex responsible for ER export, these data suggest that CNIH4 acts as a cargo‐sorting receptor, recruiting GPCRs into COPII vesicles .