title: Gβγ Binds to the Extreme C Terminus of SNAP25 to Mediate the Action of Gi/o-Coupled G Protein-Coupled Receptors.
abstract: Gi/o-coupled G protein-coupled receptors can exert an inhibitory effect on vesicle release through several G protein-driven mechanisms, more than one of which may be concurrently present in individual presynaptic terminals. The synaptosomal-associated protein of 25 kDa (SNAP25) is a key downstream effector of Gβγ subunits. It has previously been shown that proteolytic cleavage of SNAP25 by botulinum toxin A reduces the ability of Gβγ to compete with the calcium sensor synaptotagmin 1 (Syt1) for binding to SNAP25 in a calcium-dependent manner. These truncated SNAP25 proteins sustain a low level of exocytosis but are unable to support serotonin-mediated inhibition of exocytosis in lamprey spinal neurons. Here, we generate a SNAP25 extreme C-terminal mutant that is deficient in its ability to bind Gβγ while retaining normal calcium-dependent Syt1 binding to soluble N-ethylmaleimide attachment protein receptor (SNARE) and vesicle release. The SNAP25Δ3 mutant, in which residue G204 is replaced by a stop codon, features a partial reduction in Gβ1γ2 binding in vitro as well as a partial reduction in the ability of the lamprey 5-hydroxytryptamine1b-type serotonin receptor to reduce excitatory postsynaptic current amplitudes, an effect previously shown to be mediated through the interaction of Gβγ with SNAP25. Syt1 calcium-dependent binding to SNAP25Δ3 was reduced by a small extent compared with the wild type. We conclude that the extreme C terminus of SNAP25 is a critical region for the Gβγ-SNARE interaction.
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (Z.Z., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois, Chicago, Illinois (S.R., S.A.).
authors.forename: Zack
authors.initials: Z
authors.lastname: Zurawski
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (Z.Z., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois, Chicago, Illinois (S.R., S.A.).
authors.forename: Shelagh
authors.initials: S
authors.lastname: Rodriguez
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (Z.Z., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois, Chicago, Illinois (S.R., S.A.).
authors.forename: Karren
authors.initials: K
authors.lastname: Hyde
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (Z.Z., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois, Chicago, Illinois (S.R., S.A.).
authors.forename: Simon
authors.initials: S
authors.lastname: Alford
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee (Z.Z., K.H., H.E.H.); and Department of Biological Sciences, University of Illinois, Chicago, Illinois (S.R., S.A.) heidi.hamm@vanderbilt.edu.
authors.forename: Heidi E
authors.initials: HE
authors.lastname: Hamm
mesh_terms: D000818:Animals; D002417:Cattle; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry / Q000502:physiology*; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D048428:Petromyzon; D011485:Protein Binding / Q000502:physiology; D017433:Protein Structure, Secondary; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; C494209:Snap25 protein, mouse; D050825:Synaptosomal-Associated Protein 25; D019206:GTP-Binding Protein alpha Subunits, Gi-Go