G protein βγ directly regulates SNARE protein fusion machinery for secretory granule exocytosis

Abstract

The activation of G protein-coupled receptors (GPCRs) can result in an inhibition of Ca(2+)-dependent hormone and neurotransmitter secretion. This has been attributed in part to G protein inhibition of Ca(2+) influx. However, a frequently dominant inhibitory effect, of unknown mechanism, also occurs distal to Ca(2+) entry. Here we characterize direct inhibitory actions of G protein betagamma (Gbetagamma) on Ca(2+)-triggered vesicle exocytosis in permeable PC12 cells. Gbetagamma inhibition was rapid (<1 s) and was attenuated by cleavage of synaptosome-associated protein of 25 kD (SNAP25). Gbetagamma bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, and binding was reduced to SNARE complexes containing cleaved SNAP25 or by Ca(2+)-dependent synaptotagmin binding. Here we show inhibitory coupling between GPCRs and vesicle exocytosis mediated directly by Gbetagamma interactions with the Ca(2+)-dependent fusion machinery.

Molecular mechanisms that control initiation and termination of physiological depolarization-evoked transmitter release

“…Because of the rapidity of release (a few milliseconds), it is unlikely that second messengers are involved in control of the tonic block. Rapid control could potentially be achieved (i) by GPCRs targeting Ca 2ϩ channels (31,34,41), (ii) by GPCRs targeting the release machinery via G ␤ ␥ (11,12,42), or (iii) by a rapid voltagedependent shift of the GPCR affinity and a direct interaction of the GPCR itself with the release machinery (14,15).…”

“…G ␤ ␥ was shown to inhibit transmitter release (11,12) without affecting Ca 2ϩ currents. It is thus possible that PTX exerts its effects by reducing the levels of G ␤ ␥.…”

“…More importantly, the mechanism underlying the tonic block is not known even for the cholinergic synapse. It could be produced by G ␤ ␥ because it inhibits release by interacting with proteins of the release machinery (11,12). Alternatively, it could be achieved by a direct interaction of M 2 R with voltage-dependent Ca 2ϩ channels (13), or it could result from the transmitter-bound M 2 R directly interacting with the release proteins (14,15).…”

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

“…Evidence for the functional significance of this effect in neurons is still scant, although it is known that G protein regulation of N-type channels in chick calyces is altered upon treatment with botulinum toxin C1, 73 and Gβγ subunits have been shown to regulate neurotransmission with their interactions with SNARE complexes. 74 A third regulatory modality linked to the synprint motif is subcellular targeting of the channel. Work from Mochida et al 50,75 shows that the presence of the synaptic protein interaction site is an important determinant of subcellular targeting of P/Q-type channels.…”

G protein βγ-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties

“…We have demonstrated that GPCR activation leads to G␤␥ binding to the C-terminal of SNAP-25 in the SNARE complex (2,9,26) at a site coincident with synaptotagmin-SNARE complex binding. Modification of synaptotagmin-SNARE complex interactions can cause incomplete fusion of dense core vesicles (27,28), which may modify neurotransmitter release (17).…”