G protein betagamma directly regulates SNARE protein fusion machinery for secretory granule exocytosis.
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.
Blackmer
Trillium
T
Department of Biological Sciences, University of Illinois at Chicago, 840 West Taylor Street, Chicago, Illinois 60607, USA.
Larsen
Eric C
EC
Bartleson
Cheryl
C
Kowalchyk
Judith A
JA
Yoon
Eun-Ja
EJ
Preininger
Anita M
AM
Alford
Simon
S
Hamm
Heidi E
HE
Martin
Thomas F J
TF
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005136:Eye Proteins / Q000494:pharmacology; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits / Q000494:pharmacology / Q000502:physiology*; D044388:GTP-Binding Protein gamma Subunits / Q000494:pharmacology / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000494:pharmacology; D009433:Neural Inhibition / Q000187:drug effects; D009638:Norepinephrine / Q000378:metabolism; D016716:PC12 Cells; D010750:Phosphoproteins / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011930:Reaction Time / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism*; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D013997:Time Factors; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D020555:GTP-Binding Protein Regulators; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; C065631:phosducin; D050857:Synaptotagmins; D002118:Calcium; D009638:Norepinephrine