Pertussis toxin-sensitive G proteins are transported toward synaptic terminals by fast axonal transport.

Abstract

We rind that half of the pertussis toxinsensitive guanine nucleotide-binding protein (G protein) in the squid (Loligo pealei) giant axon is cytoplasmic and that this species of G protein is intermediate in size between the two forms present in axolemma. This G protein is transported toward synaptic terminals at 44 mm/day. Moreover, these data are consistent with there being two additional steps leading to the maturation of G proteins: (i). association with and transport on intracellular organelles and (ii) modification at the time of transfer to the plasmalemma resulting in a molecular weight shift. Since the other two components of G proteinmediated signal transduction pathways, receptors and effector enzymes, are known to be delivered to the synaptic terminals by fast axonal transport, our rindings introduce the possibility that these three macromolecules are assembled as a complex in the cell body and delivered together to the plasma membrane of the axon and synaptic terminals.G proteins are heterotrimeric, membrane-associated proteins that couple plasma membrane receptors to effector enzymes (1). In nerve cells these signal transduction elements are found in synaptic terminals and growth cones (2-5). The receptors and effectors are integral membrane proteins and, thus, are synthesized in the rough endoplasmic reticulum and then transported to terminals on vesicles by fast axonal transport (6-10). G protein mRNAs do not encode hydrophobic membrane-spanning segments (11), however, so it is unclear when they become associated with membranes and how they are translocated to the plasma membrane. Nonetheless, the subclass of G proteins sensitive to pertussis toxin (PTX) must be transported by synaptic terminals because they, and the receptors that they are known to be coupled with, are located there. If G proteins are indeed carried on cytoplasmic transport vesicles, we should be able to detect them in the axoplasm as they travel from the cell body to the nerve terminal. The approach that we have used to determine whether PTX-sensitive G proteins are associated with transport vesicles is to block axonal transport by focally cooling the axon. When we focally cool (cold block) a short segment of the squid giant axon to 40C, vesicles being transported in the anterograde direction accumulate on the proximal side of the cold block, whereas retrogradely transported vesicles build up on the distal side (12). Segments of axon from both sides of the cooled axon segment can be isolated and assayed for G proteins using PTX-catalyzed ADP-ribosylation and immunoblotting with G protein-specific antibodies. G proteins sensitive to PTX constitute a subclass known to interact with receptors and effectors that are important in controlling neuronal function (13). Tris HCl, pH 7.5/150 mM NaCl/3 mM EDTA/1 mM EGTA, 5 mM 2-mercaptoethanol/5 mM benzamidine) for every centimeter of axon. Extrusion of axoplasm and the recovery of axoplasm and axon sheath (axolemma) were performed by the method described by Lasek (15). Axoplasmic h...

GTP gamma S inhibits organelle transport along axonal microtubules.

“…In experiments described here, various probes for GTP-binding proteins, and broad spectrum inhibitors of protein kinases or phosphatases were added to the perfusion buffer, and the effects on organelle transport velocity were monitored by quantitative video microscopy. Prior studies have demonstrated the presence of such regulatory proteins in squid axoplasm (22,37,82). The results of the current studies imply that fast axonal transport is modulated by cycles of GTP hydrolysis and nucleotide exchange by one or more species of GTP-binding protein.…”

“…Proteins that are known to bind guanine nucleotides and have been implicated in regulation of membrane traffic include numerous heterotrimeric G-proteins and small GTP-binding proteins, as well as dynamin. Pertussis toxin-sensitive G-proteins are known to be present in squid axoplasm (82), and to test whether the effects of GTPTS on fast axonal transport might be due to these or other G-proteins, axons were perfused with AIF4-. This salt, which is formed by mixing low concentrations (1-100 #M) of A1CI3 with higher concentrations (5-30 mM) of KF or NaF, mimics the effects of GTPTS on all known heterotrimeric G-proteins, but does not affect small GTP-binding proteins (34,74).…”

Nerve growth factor changes G protein levels and localization in PC12 cells

“…We do not know the mechanisms that govern this translocation. In squid growth axons, G proteins that are pertussis toxin substrates can be transported distally by fast axonal transport (Vogel et al, 1991). In some PC12 cells that have not yet differentiated or are in early stages of response to NGF, a,, immunoreactivity is localized to filopodial protrusions or developing processes.…”

Retrograde Axonal Transport of the α‐Subunit of the GTP‐binding Protein Gz in Mouse Sciatic Nerve: a Potential Pathway for Signal Transduction In Neurons

“…For this reason, we have examined whether such a transduction mechanism may exist in the neuron, whereby GTPbinding proteins may also be activated to leave the cell membrane to become stable second messengers transported from the axon tip to the cell body. A recent report has shown the orthograde movement of pertussis toxin substrates in squid giant axon (Vogel et al, 1991).…”