SummaryEndocytic trafficking is a critical mechanism for cells to decode complex signaling pathways, including those activated by G-protein-coupled receptors (GPCRs). Heterogeneity in the endosomal network enables GPCR activity to be spatially restricted between early endosomes (EEs) and the recently discovered endosomal compartment, the very early endosome (VEE). However, the molecular machinery driving GPCR activity from the VEE is unknown. Using luteinizing hormone receptor (LHR) as a prototype GPCR for this compartment, along with additional VEE-localized GPCRs, we identify a role for the adaptor protein APPL1 in rapid recycling and endosomal cAMP signaling without impacting the EE-localized β2-adrenergic receptor. LHR recycling is driven by receptor-mediated Gαs/cAMP signaling from the VEE and PKA-dependent phosphorylation of APPL1 at serine 410. Receptor/Gαs endosomal signaling is localized to microdomains of heterogeneous VEE populations and regulated by APPL1 phosphorylation. Our study uncovers a highly integrated inter-endosomal communication system enabling cells to tightly regulate spatially encoded signaling.

SummarySpatial control of G-protein-coupled receptor (GPCR) signaling, which is used by cells to translate complex information into distinct downstream responses, is achieved by using plasma membrane (PM) and endocytic-derived signaling pathways. The roles of the endomembrane in regulating such pleiotropic signaling via multiple G-protein pathways remain unknown. Here, we investigated the effects of disease-causing mutations of the adaptor protein-2 σ subunit (AP2σ) on signaling by the class C GPCR calcium-sensing receptor (CaSR). These AP2σ mutations increase CaSR PM expression yet paradoxically reduce CaSR signaling. Hypercalcemia-associated AP2σ mutations reduced CaSR signaling via Gαq/11 and Gαi/o pathways. The mutations also delayed CaSR internalization due to prolonged residency time of CaSR in clathrin structures that impaired or abolished endosomal signaling, which was predominantly mediated by Gαq/11. Thus, compartmental bias for CaSR-mediated Gαq/11 endomembrane signaling provides a mechanistic basis for multidimensional GPCR signaling.

Agonist stimulation of G protein–coupled receptors (GPCRs) typically leads to phosphorylation of GPCRs and binding to multifunctional proteins called β-arrestins (βarrs). The GPCR–βarr interaction critically contributes to GPCR desensitization, endocytosis, and downstream signaling, and GPCR–βarr complex formation can be used as a generic readout of GPCR and βarr activation. Although several methods are currently available to monitor GPCR–βarr interactions, additional sensors to visualize them may expand the toolbox and complement existing methods. We have previously described antibody fragments (FABs) that recognize activated βarr1 upon its interaction with the vasopressin V2 receptor C-terminal phosphopeptide (V2Rpp). Here, we demonstrate that these FABs efficiently report the formation of a GPCR–βarr1 complex for a broad set of chimeric GPCRs harboring the V2R C terminus. We adapted these FABs to an intrabody format by converting them to single-chain variable fragments (ScFvs) and used them to monitor the localization and trafficking of βarr1 in live cells. We observed that upon agonist simulation of cells expressing chimeric GPCRs, these intrabodies first translocate to the cell surface, followed by trafficking into intracellular vesicles. The translocation pattern of intrabodies mirrored that of βarr1, and the intrabodies co-localized with βarr1 at the cell surface and in intracellular vesicles. Interestingly, we discovered that intrabody sensors can also report βarr1 recruitment and trafficking for several unmodified GPCRs. Our characterization of intrabody sensors for βarr1 recruitment and trafficking expands currently available approaches to visualize GPCR–βarr1 binding, which may help decipher additional aspects of GPCR signaling and regulation.

Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.

During clathrin mediated endocytosis (CME), the concerted action of dynamin and its interacting partners drives membrane scission. Essential interactions occur between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 (SH3) of various proteins including amphiphysins. Here we show that multiple SH3 domains must bind simultaneously to dynPRD through three adjacent motifs for dynamin’s efficient recruitment and function. First, we show that mutant dynamins modified in a single motif, including the central amphiphysin SH3 (amphSH3) binding motif, partially rescue CME in dynamin triple knock-out cells. However, mutating two motifs largely prevents that ability. Furthermore, we designed divalent dynPRD-derived peptides. These ligands bind multimers of amphSH3 with >100-fold higher affinity than monovalent ones in vitro. Accordingly, dialyzing living cells with these divalent peptides through a patch-clamp pipette blocks CME much more effectively than with monovalent ones. We conclude that dynamin drives vesicle scission via multivalent interactions in cells.

This protocol describes a procedure for live-cell imaging of endocytic events in cultured cells using a pH-sensitive fluorophore and fast extracellular pH changes. A Matlab-based analysis pipeline is provided to facilitate automated data processing.TWEET A fully automated protocol for imaging endocytosis in cultured living cells COVER TEASER Imaging endocytic vesicle scission in cultured living cells

The chemokine prokineticin 2 (PK2) activates its cognate G protein-coupled receptor (GPCR) PKR2 to elicit various downstream signaling pathways involved in diverse biological processes. Many GPCRs undergo dimerization that can modulate a number of functions including membrane delivery and signal transduction. The aim of this study was to elucidate the interface of PKR2 protomers within dimers by analyzing the ability of PKR2 transmembrane (TM) deletion mutants to associate with wild type (WT) PKR2 in yeast using co-immunoprecipitation and mammalian cells using bioluminescence resonance energy transfer. Deletion of TMs 5-7 resulted in a lack of detectable association with WT PKR2, but could associate with a truncated mutant lacking TMs 6-7 (TM1-5). Interestingly, TM1-5 modulated the distance, or organization, between protomers and positively regulated Gαs signaling and surface expression of WT PKR2. We propose that PKR2 protomers form Type II dimers involving TMs 4 and 5, with a role for TM5 in modulation of PKR2 function. We have carefully considered the reviewers' comments and suggestions and extensively edited the manuscript to address them, including addition of new data. In particular, we have included further controls to confirm that BRET-tagged receptors are functional and inclusion of BRET analysis in both HEK and CHO cells. We have also made improvements to the text in order to ensure a clearer understanding of the methodology and results. We now believe that we have fully addressed all of the concerns raised by the Reviewers and, therefore, wish to submit the revised manuscript for your consideration.Specific responses to comments together with descriptions of new data added and changes made in the text and figures, are detailed in the attached "Response to Reviewers." We believe that the revised manuscript is substantially improved, and we thank the reviewers for their positive comments and for aiding with refinement and clarity of this study.Thank you again for your consideration, and we hope that you will now find our work acceptable for publication. Yours sincerely, Aylin Hanyaloglu and Rosella MieleCover Letter Response to ReviewersReviewer #1: There is one concern: the backgrounds of the images in figure 2 are different--which suggests either differential processing or alteration of the contrast.We would like to thank the reviewer for their overall positive comments. In regards to Figure 2 we would like to assure the reviewer there was no post-acquisition modification of the figures. The development of film in the laboratory in Rome is carried out without an automated film processor but incubation in individual baths of developer, which can result in background differences of the films. The original films were presented as photographed images in the original submission thus we have now scanned the original films and presented them in grey-scale to improve image clarity (please see new Figure 2) but again we would like to emphasize that no alterations of the contrast were made. We hope th...

Follicle-stimulating hormone receptor (FSHR) is a G protein-coupled receptor (GPCR) with pivotal roles in reproduction. One key mechanism dictating the signal activity of GPCRs is membrane trafficking. After binding its hormone FSH, FSHR undergoes internalization to very early endosomes (VEEs) for its acute signaling and sorting to a rapid recycling pathway. The VEE is a heterogeneous compartment containing the Adaptor Protein Phosphotyrosine Interacting with Pleckstrin homology Domain and Leucine Zipper 1 (APPL1) with distinct functions in regulating endosomal Gαs/cAMP signaling and rapid recycling. Low molecular weight (LMW) allosteric FSHR ligands were developed for use in assisted reproductive technology yet could also provide novel pharmacological tools to study FSHR. Given the critical nature of receptor internalization and endosomal signaling for FSHR activity, we assessed whether these compounds exhibit differential abilities to alter receptor endosomal trafficking and signaling within the VEE. Two chemically distinct LMW agonists (benzamide, termed B3 and thiazolidinone, termed T1) were employed. T1 was able to induce a greater level of cAMP than FSH and B3. As cAMP signaling drives gonadotrophin hormone receptor recycling, rapid exocytic events were evaluated at single event resolution. Strikingly, T1 was able to induce a 3-fold increase in recycling events compared to FSH and two-fold more compared to B3. As T1-induced internalization was only marginally greater, the dramatic increase in recycling and cAMP signaling may be due to additional mechanisms. All compounds exhibited a similar requirement for receptor internalization to increase cAMP and proportion of FSHR endosomes with active Gαs, suggesting regulation of cAMP signaling induced by T1 may be altered. APPL1 plays a central role for GPCRs targeted to the VEE, and indeed, loss of APPL1 inhibited FSH-induced recycling and increased endosomal cAMP signaling. While T1-induced FSHR recycling was APPL1-dependent, its elevated cAMP signaling was only partially increased following APPL1 knockdown. Unexpectedly, B3 altered the dependence of FSHR to APPL1 in an opposing manner, whereby its endosomal signaling was negatively regulated by APPL1, while B3-induced FSHR recycling was APPL1-independent. Overall, FSHR allosteric compounds have the potential to re-program FSHR activity via altering engagement with VEE machinery and also suggests that these two distinct functions of APPL1 can potentially be selected pharmacologically.