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First published online January 27, 2006
doi: 10.1242/10.1242/jcs.02766

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The neuropeptide head activator is a high-affinity ligand for the orphan G-protein-coupled receptor GPR37

¹ Zentrum für Molekulare Neurobiologie Hamburg, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
² Klinik und Poliklinik für Neurologie, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
³ Institut für Angewandte Physiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
⁴ RIKEN Brain Science Institute, Saitama 351-0198, Japan

View larger version (80K): [in a new window]   Fig. 1. GPR37 is expressed at the cell surface of COS-7 cells and internalises after HA treatment. (A-I) COS-7 cells were transfected with GPR37 with (B,D-I) or without (A,C) FLAG tag, immunostained with anti-GPR37 antibody (A,C,D) or with anti-FLAG antibody (B,E-I) and visualised with alkaline phosphatase-coupled secondary antibodies for light microscopy (A) or with Cy3-coupled antibodies for confocal analysis (B-I). Cells were permeabilised (perm) by fixation with 1% acetic acid in ethanol and by washing with Triton X-100, except in D, where living cells were incubated with the primary antibody before fixation (non-perm) to show surface staining. (E-I) COS-7 cells 48 hours after transfection with GPR37-FLAG were treated at 37°C with 2 nM HA for 0, 10, 20, 30 and 60 minutes, respectively, and immunostained with anti-FLAG antibody.

View larger version (63K): [in a new window]   Fig. 2. Inducible, stable expression of GPR37 prevents aggregate formation. (A) CHO-K1, COS-7 and HEK-293 cells were transiently transfected with GPR37, and membrane fractions were assayed by immunoblotting (IB) with anti-GPR37 antibody (-GPR37). (B) COS-7 cells transiently transfected with GPR37-FLAG were cell-surface biotinylated, and the solubilised membrane fraction was immunoprecipated (IP) with anti-FLAG antibody (-FLAG) and visualised after immunoblotting with avidin. (C) GPR37 was introduced stably into the flip-in cell line HEK-T-REx, where GPR37 expression is inducible by doxycycline (DOX). Membrane fractions were subjected to western blotting with anti-GPR37 antibody (-GPR37) with (first lane) and without (second lane) induction for 24 hours with doxycycline. (D-I) HEK-T-REx-GPR37 cells with (D,F,H,I) and without (E,G) doxycycline induction for 24 hours were immunostained with anti-GPR37(R2) antibody after permeabilisation (D,E) and with anti-GPR37 antibody without permeabilisation (F-I). (H,I) HEK-T-REx-GPR37 cells were treated with 2 nM HA for 0 and 10 minutes at 37°C, respectively, fixed with 2% formaldehyde for 10 minutes and subsequently immunostained with anti-GPR37 antibody.

View larger version (115K): [in a new window]   Fig. 3. HA colocalises with and binds to GPR37. (A-D) Interaction of HA and GPR37 analysed by FRET. Shown is a typical example of FRET between HA and an extracellular epitope of GPR37. COS-7 cells transiently transfected with GPR37 were treated with 2 nM HA for 20 minutes on ice to prevent internalisation, followed by incubation for 20 minute on ice with the antiserum against HA (-HA). After fixation with 4% formaldehyde in PBS, cells were immunostained with anti-GPR37 antibody (-GPR37). GPR37 immunoreactivity was visualised with Cy3 (A,B) and that of HA with Alexa Fluor 488 (C,D). (A) The Cy3 signal (GPR37) is shown after excitation at 568 nm. (B) A discrete area was photobleached using intense 568 nm laser. The Alexa Fluor 488 signal (HA) after excitation at 488 nm is shown before (C) and after (D) photobleaching. In this example, the Alexa Fluor 488 signal was increased by 18%. (E-G) The neuroblastoma cell line NH15-CA2 was used as a positive control to show specific Cy3B-HA binding to endogenous HA receptors. (E) NH15-CA2 cells endogenously express GPR37, as visualised with anti-GPR37(R1) antibody [-GPR37(R1)]. (F,G) Binding is optimal at 150 nM of Cy3B-HA after incubation for 10 minutes at 37°C (Cy3B-HA) and is inhibited by pretreatment for 50 minutes at 37°C with 100 nM unlabelled, monomerised HA (Cy3B-HA comp). (H-J) HEK-T-REx-GPR37 cells bound Cy3B-HA only after GPR37 induction with doxycycline (±DOX), and binding was competed with unlabelled HA (+DOX comp).

View larger version (38K): [in a new window]   Fig. 4. HA stimulates Ca2+ mobilisation in CHO-K1 cells stably transfected with GPR37-FLAG, G16 and apoaequorin. (A) The Ca2+-bioluminescence response was measured at 469 nm and is expressed in relative light units (RLU), from which the medium response was subtracted. Values are given as means ± s.d. CHO-G16-AEQ cells stably expressing GPR37-FLAG (CHO-GPR37) responded with an EC50 value of 3.3 nM; the endogenous response of CHO-G16-AEQ cells (CHO) resulted in an EC50 value of 11 nM. Data show representative results of three independent experiments. (B) CHO-GPR37 (upper panel) and CHO cells (lower panel) reacted with anti-GPR37(R1) antibody [-GPR37(R1)], a polyclonal antiserum produced against the conserved intracellular C-tail. (C) Western blot analysis of membrane fractions confirmed an increased expression of GPR37 in transfected cells. The mouse hippocampal cell line HT22, expressing GPR37 endogenously, was used as a positive control.

View larger version (14K): [in a new window]   Fig. 5. HA is a high-affinity ligand for GPR37 expressed in frog oocytes. (A) Currents induced by 100 nM HA were recorded from Xenopus oocytes injected with cRNAs coding for GPR37 and for GIRK1/2. Stimulation with medium served as control. (B) The current increase was dependent on HA concentration. Dose-response curves for a HA-induced increase in GIRK1/2-mediated inward currents were normalised against maximal currents obtained for each oocyte. Current increases were averaged over four oocytes prepared and injected on the same day. The values represent means ± s.d. Data are representative of several independent experiments.

View larger version (21K): [in a new window]   Fig. 6. GPR37 mediates HA signalling to stimulate mitosis. (A) HEK-T-REx-GPR37 cells were treated with and without doxycycline for 24 hours. Incubation with 2 nM HA for 1.7 hours led to an increase of cells in mitosis after induction of GPR37 expression. Immunostaining of cells with anti-phospho-histone H3 (1:1000) was used to determine cells in mitosis. 6x350 cells were counted, and the percentage of stained mitotic cells is given as means ± s.d. (B) Membrane currents were measured in the perforated patch configuration at a holding potential of -80 mV. Treatment with 1 nM HA induced an increase in membrane currents in COS-7 cells transiently expressing GPR37 (COS-GPR37), but not in mock-injected cells (COS-Mock). Membrane currents activated by HA in HEK-T-REx-GPR37 cells were blocked by application of 1 mM La3+ or 10 µM SKF. (C) Membrane-current densities were recorded from mock- and GPR37-transfected COS-7 cells. HA signal transduction was inhibited by pretreating cells for 2-3 hours with 200 ng ml-1 pertussis toxin (PTX), for 30-60 minutes with 100 nM wortmannin (WORT), or 30 µM KN93 (KN). Each symbol represents one cell measured in the whole-cell (filled symbols) or the perforated patch (open symbols) configuration

View larger version (22K): [in a new window]   Fig. 7. Scheme of the signalling pathway from HA through GPR37 to stimulate mitosis. After binding of HA to GPR37 with or without help of the coreceptor SorLA, a pertussis-toxin-sensitive inhibitory G protein (Gi) is activated, which interacts through the phosphoinositide 3-kinase (PI3K) and the calcium-calmodulin dependent kinase II (CaMK) with a Ca2+ channel of the transient receptor potential family (TRPV2-like). The resulting Ca2+ influx activates a Ca2+-dependent K+ channel of the small and intermediate conductance family (SK4-like), leading to hyperpolarisation, which is a prerequisite for cells to enter mitosis. Dashed lines indicate hypothetical pathways.