Axonal Targeting of the 5-HT1B Serotonin Receptor Relies on Structure-Specific Constitutive Activation Damien Carrel, Anne Simon, Michel Emerit, Isabelle Rivals, Christophe Leterrier, Marc Biard, Michel Hamon, Michèle Darmon, Zsolt Lenkei To cite this version: Damien Carrel, Anne Simon, Michel Emerit, Isabelle Rivals, Christophe Leterrier, et al.. Axonal Targeting of the 5-HT1B Serotonin Receptor Relies on Structure-Specific Constitutive Activation. Traffic, Wiley, 2011, 12 (11), pp.1501-1520. 10.1111/j.1600-0854.2011.01260.x. hal-01701553 HAL Id: hal-01701553 https://hal.archives-ouvertes.fr/hal-01701553 Submitted on 20 Apr 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Traffic 2011; 12: 1501–1520 © 2011 John Wiley & Sons A/S doi:10.1111/j.1600-0854.2011.01260.x Axonal Targeting of the 5-HT1B Serotonin Receptor Relies on Structure-Specific Constitutive Activation Damien Carrel1,2,3,#, Anne Simon3,#,MichelB. into two functionally different main sub-domains, the Emerit1,2, Isabelle Rivals4, Christophe axonal and somatodendritic compartments (1,2). Polarized sorting mechanisms to the axonal and somatodendritic Leterrier3,5,MarcBiard3, Michel Hamon1,2, surface have been studied using model proteins, mainly 1,2,∗,† 3,∗,† Michele` Darmon and Zsolt Lenkei cell adhesion molecules (3–7). Axonal targeting of these proteins relies either on trans-Golgi network (TGN)-based sorting via still poorly understood signals and machinery, 1FacultedeM´ edecine´ Pierre et Marie Curie, Universite´ or on endocytic removal of misplaced proteins from the Pierre et Marie Curie, Site Pitie-Salp´ etriˆ ere,` Paris 6 2INSERM, U894, Paris F-75013, France somatodendritic plasma membrane, followed by degra- 3Laboratoire de Neurobiologie, CNRS UMR7637, dation or by signal-mediated recycling to the axon from ESPCI-ParisTech, Paris F-75005, France somatodendritic endosomes (reviewed in 8). 4Equipe´ de Statistique Appliquee,´ ESPCI-ParisTech, Paris F-75005, France The superfamily of G-protein coupled receptors (GPCRs) 5Current address: INSERM U641 - Universitedela´ regroups hundreds of sensory proteins, which regulate Mediterran´ ee,´ F-13916 Marseille, France vital physiological functions and represent eminent thera- *Corresponding authors: Michele` Darmon, peutic targets. GPCRs are highly dynamic, composed of a [email protected] and Zsolt Lenkei, zsolt.lenkei@ core of relatively rigid transmembrane alpha helices con- espci.fr nected by flexible loops, and display a conformational equi- #,† These authors contributed equally to this work. librium at the plasma membrane at steady state. Evidence from both functional and biophysical studies suggests that By analogy to other axonal proteins, transcytotic delivery GPCRs permanently sample multiple conformations but following spontaneous endocytosis from the somatoden- most are held in a basal conformational equilibrium by dritic membrane is expected to be essential for polar- intervening loops and non-covalent intramolecular inter- ized distribution of axonal G-protein coupled receptors actions (9). Agonist and partial agonist ligands shift the (GPCRs). However, possible contribution from constitu- equilibrium toward activated states, whereas inverse ago- tive activation, which may also result in constitutive nists shift the equilibrium toward the inactive states. GPCR endocytosis, is poorly known. Using two closely Adoption of an activated state leads to the rearrangement related but differentially distributed serotonin receptors, here we demonstrate higher constitutive activation and of the cytoplasmic side of the receptor, leading to mobi- lization of intracellular signaling pathways mainly through spontaneous endocytosis for the axonal 5-HT1BR, as compared to the somatodendritic 5-HT1AR, both in non- a cognate heterotrimeric G-protein and also to interac- neuronal cells and neurons. Activation-dependent consti- tion with different other protein partners, resulting in tutive endocytosis is crucial for axonal targeting, because phosphorylation-dependent desensitization and ultimately inverse-agonist treatment, which prevents constitutive in clathrin-mediated endocytosis (10). activation, leads to atypical accumulation of newly syn- thesized 5-HT1BRs on the somatodendritic plasma mem- Numerous GPCRs exhibit polarized distribution on the brane. Using receptor chimeras composed of different neuronal plasma membrane. This polarization critically domains from 5-HT Rand5-HT R, we show that the 1A 1B determines the physiological effects that follow their complete third intracellular loop of 5-HT1BR is neces- sary and sufficient for constitutive activation and efficient activation. While several determinants of dendritic local- axonal targeting, both sensitive to inverse-agonist treat- ization have been recently identified, the mechanisms ment. These results suggest that activation and targeting responsible for axonal GPCR targeting and localization of 5-HT1BRs are intimately interconnected in neurons. remain for the most part unknown (8). We recently pro- posed constitutive somatodendritic endocytosis followed Key words: axon, constitutive activity, polarized distribu- by transcytotic delivery to the axonal plasma membrane tion, targeting, third intracellular loop, 7-transmembrane for an axonal GPCR, the type-1 cannabinoid receptor (11). receptor, p11 However, the involvement of steady-state receptor acti- vation, an important regulator of constitutive GPCR Received 2 July 2010, revised and accepted for publication endocytosis (12–17), is still under debate (18); thus, the 28 July 2011, uncorrected manuscript published online 1 relation between axonal targeting and receptor conforma- August 2011, published online 6 September 2011 tions triggering activation-dependent endocytosis is poorly known. The highly polarized neuronal plasma membrane is divided Serotonin (5-HT) receptors play key roles in cognition, by the axon initial segment, acting as a diffusion barrier, mood and pathological situations such as psychosis www.traffic.dk 1501 Carrel et al. E A B C F D G H K I J Figure 1: Legend on next page. 1502 Traffic 2011; 12: 1501–1520 Constitutive Endocytosis Targets 5-HT1BR to Axons by regulating transmitter release, synaptic integrity and composed of different domains of 5-HT1AR and 5-HT1BR, neural plasticity (19,20). The 5-HT1A and 5-HT1B serotonin we have focused our investigation on the specific role of receptors are two closely related GPCRs, which are the third intracellular loop of 5-HT1BR. coupled to Gi/o proteins and mobilize similar signaling pathways (21). In neurons, both have autoreceptor func- tions but differ in the mechanism by which they control Results serotonin release. Whereas 5-HT1AR modulates the fir- ing of serotonergic neurons in the raphe nuclei (22), the Axonal localization of 5-HT1BRisaresult 5-HT1BR participates in a local control of serotonin release of constitutive somatodendritic endocytosis from axon terminals in their projection areas (19). This In neurons, both in vitro and in vivo data indicate functional difference is governed by differential subcellular that 5-HT1BRs are localized at the axonal plasma mem- distribution of these two receptors in neurons. Investiga- brane, whereas 5-HT1AR are mainly localized at plasma tion by specific radioligand binding, in situ hybridization, membrane of soma and dendrites (27,29). We have immunohistochemistry and immuno-electron microscopy directly compared the surface localization of transfected showed that 5-HT1AR is mainly localized on the soma- 5-HT1AR and 5-HT1BR, tagged with a short, eight-amino- todendritic plasma membrane, whereas the 5-HT1BR acid-long Flag-epitope at the extracellular N-terminus, in is located in the plasma membrane of unmyelinated low-density primary cultures of living hippocampal neu- axons, mostly outside of terminals and synaptic differ- rons (Figure 1A–D). Receptor surface localization was entiations, and is also found in cytoplasmic vesicles in analyzed using two quantification methods. First, the the somatodendritic region (23–27). Previously, a com- ratio of axonal versus somatodendritic surface label- parative study of 5-HT1BR and 5-HT1AR targeting using ing (A/D ratio) was measured for individual transfected chimeras of the two serotonin receptors showed that the neurons. In addition, transfected neurons were system- third intracellular loop (I3) is important for the axonal tar- atically searched and classified according to the distribu- geting of 5-HT1BR (28,29). However, the mechanism by tion of surface receptor by an investigator blind to the which the third intracellular loop regulates axonal target- experimental condition (see Materials and Methods for ing of 5HT1BR remain unknown. More generally, it is not details). Both approaches clearly showed that while there known whether 5HT1BR is targeted to the axonal surface is a constant proportion of uniformly labeled neurons, through a direct, post-TGN sorting mechanism or by the 5-HT1BR is predominantly localized to the axonal surface, indirect, somatodendritic-endocytosis-driven