Neural circuit assembly in the brain requires precise establishment of synaptic connections, but the mechanisms of synapse assembly remain incompletely understood. Latrophilins are postsynaptic adhesion-GPCRs that engage in trans-synaptic complexes with presynaptic teneurins and FLRTs. In mouse CA1-region neurons, Latrophilin-2 and Latrophilin-3 are essential for formation of entorhinal-cortex-derived and Schaffer-collateral-derived synapses, respectively. However, it is unknown whether latrophilins function as GPCRs in synapse formation. Here, we show that Latrophilin-2 and Latrophilin-3 exhibit constitutive GPCR activity that increases cAMP levels, which was blocked by a mutation interfering with G-protein and arrestin interactions of GPCRs. The same mutation impaired the ability of Latrophilin-2 and Latrophilin-3 to rescue the synapse-loss phenotype in Latrophilin-2 and Latrophilin-3 knockout neurons in vivo. Our results suggest that Latrophilin-2 and Latrophilin-3 require GPCR signaling in synapse formation, indicating that latrophilins promote synapse formation in the hippocampus by activating a classical GPCR-signaling pathway.
All raw numerical data within the study has been submitted together with the manuscript.
- Richard Sando
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All procedures strictly conformed to National Institutes of Health Guidelines for the Care and Use of Laboratory Mice and were approved by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC) and institutional animal care and use committee (IACUC). The animal protocol #20787 was approved by Stanford University APLAC and IACUC. All surgeries were performed under Avertin anesthesia and buprenorphine analgesia, and every effort was made to minimize suffering, pain and distress.
- Graeme W Davis, University of California, San Francisco, United States
© 2021, Sando & Südhof
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1’s Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1’s helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions via its BRCT and ARD motifs, binds SLF2 analogously to SIMC1 and forms a separate Nse5/6-like complex. Thus, two Nse5/6-like complexes with distinct recruitment domains control human SMC5/6 localization.
Opioid tolerance is well-described physiologically but its mechanistic basis remains incompletely understood. An important site of opioid action in vivo is the presynaptic terminal, where opioids inhibit transmitter release. This response characteristically resists desensitization over minutes yet becomes gradually tolerant over hours, and how this is possible remains unknown. Here, we delineate a cellular mechanism underlying this longer-term form of opioid tolerance in cultured rat medium spiny neurons. Our results support a model in which presynaptic tolerance is mediated by a gradual depletion of cognate receptors from the axon surface through iterative rounds of receptor endocytosis and recycling. For the μ-opioid receptor (MOR), we show that the agonist-induced endocytic process which initiates iterative receptor cycling requires GRK2/3-mediated phosphorylation of the receptor’s cytoplasmic tail, and that partial or biased agonist drugs with reduced ability to drive phosphorylation-dependent endocytosis in terminals produce correspondingly less presynaptic tolerance. We then show that the δ-opioid receptor (DOR) conforms to the same general paradigm except that presynaptic endocytosis of DOR, in contrast to MOR, does not require phosphorylation of the receptor’s cytoplasmic tail. Further, we show that DOR recycles less efficiently than MOR in axons and, consistent with this, that DOR tolerance develops more strongly. Together, these results delineate a cellular basis for the development of presynaptic tolerance to opioids and describe a methodology useful for investigating presynaptic neuromodulation more broadly.