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CB1 cannabinoid receptor is widely expressed in the central nervous system of animals from late prenatal development to adulthood. Appropriate activation and signaling of CB1 cannabinoid receptors in cortical interneurons are crucial during perinatal/postnatal ages and adolescence, when long‐lasting changes in brain activity may elicit subsequent appearance of disorders in the adult brain. Here we used an optimized immunoprecipitation protocol based on specific antibodies followed by shot‐gun proteomics to find CB1 interacting partners in postnatal rat GABAergic cortical neurons in vitro at two different stages of maturation. Besides describing new proteins associated with CB1 like dihydrolipoyllysine‐residue acetyltransferase component of pyruvate dehydrogenase complex (DLAT), fatty acid synthase (FASN), tyrosine 3‐monooxygenase/tryptophan 5‐monooxygenase activation protein zeta (YWHAZ), voltage‐dependent anion channel 1 (VDAC1), myosin phosphatase Rho‐interacting protein (MPRIP) or usher syndrome type‐1C protein‐binding protein 1 (USHBP1), we show that the signaling complex of CB1 is different between maturational stages. Interestingly, the CB1 signaling complex is enriched at the more immature stage in mitochondrial associated proteins and metabolic molecular functions, whereas at more mature stage, CB1 complex is increased in maturation and synaptic‐associated proteins. We describe also interacting partners specifically immunoprecipitated with either N‐terminal or C‐terminal CB1 directed antibodies. Our results highlight new players that may be affected by altered cannabinoid signaling at this critical window of postnatal cortical development. [ABSTRACT FROM AUTHOR]

The protein anosmin-1, coded by the KAL1 gene responsible for the X-linked form of Kallmann syndrome ( KS), exerts its biological effects mainly through the interaction with and signal modulation of fibroblast growth factor receptor 1 ( FGFR1). We have previously shown the interaction of the third fibronectin-like type 3 (Fn III) domain and the N-terminal region of anosmin-1 with FGFR1. Here, we demonstrate that missense mutations reported in patients with KS, C172R and N267K did not alter or substantially reduce, respectively, the binding to FGFR1. These substitutions annulled the chemoattraction of the full-length protein over subventricular zone ( SVZ) neuronal precursors ( NPs), but they did not annul it in the N-terminal-truncated protein (A1Nt). We also show that although not essential for binding to FGFR1, the cysteine-rich ( CR) region is necessary for anosmin-1 function and that Fn III.3 cannot substitute for Fn III.1 function. Truncated proteins recapitulating nonsense mutations found in KS patients did not show the chemotropic effect on SVZ NPs, suggesting that the presence behind Fn III.1 of any part of anosmin-1 produces an unstable protein incapable of action. We also identify the extracellular signal-regulated kinase 1/2 ( ERK1/2) pathway as necessary for the chemotropic effect exerted by FGF2 and anosmin-1 on rat SVZ NPs. [ABSTRACT FROM AUTHOR]

Anosmin-1, defective in Kallmann's syndrome, participates in the adhesion, migration and differentiation of different cell types in the CNS. Although not fully understood, the mechanisms of action of Anosmin-1 involve the interaction with different proteins, being the interaction with fibroblast growth factor receptor 1 (FGFR1) and the modulation of its signalling the best studied to date. Using glutathione-S-transferase pulldown assays we demonstrate that the FnIII.3 (Fibronectin-like type III) domain and the combination whey acidic protein- FnIII.1, but not each of them individually, interact with FGFR1. The interaction of the whey acidic protein-FnIII.1 domains is substantially reduced when the cysteine-rich region is present, suggesting a likely regulatory role for this domain. The introduction in FnIII.3 of any of the two missense mutations found in Kallmann's syndrome patients, E514K and F517L, abolished the interaction with FGFR1, what suggests an important role for these residues in the interaction. Interestingly, the chemoattraction of Anosmin-1 on rat neuronal precursors (NPs) via FGFR1 is retained by the N-terminal region of Anosmin-1 but not by FnIII.3 alone, and is lost in proteins carrying either one of the missense mutations, probably because of a highly reduced binding capacity to FGFR1. We also describe homophilic interaction Anosmin-1/Anosmin-1 via the FnIII repeats 1 and 4, and the interaction of FnIII.1 and FnIII.3 with Fibronectin and of FnIII.3 with Laminin. [ABSTRACT FROM AUTHOR]