Translocation of alpha subunits of stimulatory guanine nucleotide-binding proteins through stimulation of the prostacyclin receptor in mouse mastocytoma cells.

M Negishi,H Hashimoto,A Ichikawa

doi:10.1016/s0021-9258(18)45887-5

M Negishi, H Hashimoto + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)45887-5

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Abstract

The translocation of the alpha subunits of Gs from the membrane to the cytosol by iloprost, a stable prostacyclin analogue, was studied in mouse mastocytoma P-815 cells. In the presence of guanosine 5'-O-(thiotriphosphate) (GTP gamma S), iloprost stimulated the adenylate cyclase activity, caused the release of both 42- and 45-kDa proteins reactive with the anti Gs alpha carboxyl-terminal antibody, RM/1, from the membrane and attenuated cholera toxin-catalyzed ADP-ribosylation of the 42- and 45-kDa proteins in the membrane. The iloprost-stimulated adenylate cyclase activity and release of Gs alpha from the membrane were markedly suppressed by RM/1. Cholera toxin treatment also stimulated the adenylate cyclase activity and release of Gs alpha from the membrane, and iloprost synergistically potentiated these actions of cholera toxin. In mastocytoma cells, iloprost induced the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol, 45-kDa Gs alpha remaining in the cytosol for a longer time than 42- kDa Gs alpha. Whereas 42-kDa Gs alpha in the cytosol was eluted at the position of Mr = approximately 40,000 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 from a Superose 12 gel filtration column. In contrast, both 42- and 45-kDa Gs alpha released in vitro from the membrane by iloprost plus GTP gamma S were eluted at the position of Mr = approximately 40,000, but only 45-kDa Gs alpha was eluted at the position of Mr = approximately 120,000 when it was incubated with cytosol. These results taken together demonstrate that iloprost induces the translocation of both 42- and 45-kDa Gs alpha from the membrane to the cytosol and that only the 45-kDa Gs alpha released exists in the cytosol as a soluble complex with unidentified component(s) in mastocytoma cells.

Highlights

Pcukm4yraD2ioct-neaslaaadt-lnspsedractoianh4mtctoe5itulbi-ienlvkoarsiDdatteyyradt,e,opacRxracaioMdutnitsev-e/enc1eidyan, tlwstafahriltienoyethmzrteehcttldhyeheeceaAlsmaeaDmsneeoPmetfGim-abbrsciobarbtatriohcavnasnie4yrtey2bl.aa-ontTaxidoanhyonndelaf-dttitterlheoer4en-l-5e--asteiehenofnaftvteesepacrcteosbhcureiafebcnynougutnaehpsiaetlsbsiunlf(emgu1.ne)aIcd.Fnntdritoocomnsaoeponrfptvtrehaeaaesapritsra,htryotatdihncr0eiuon-alyptaneshrrucoahGbibcdoui-tecfrnpniotirtyrtoisc,tteahtalhielnryeeafi,&unwsnuricsbtethuciuobendnpuiiattnfolfilroety-srnof Gsa from the membrane were markedly suppressedplasma membranes (3)
Whereas 42-kDa Gscr in the cytosol was genesis of the N-myristoylation consensus sequence from a eluted at the position of M, = -40,000, 45-kDa Gsa was eluted at the position of M, = -120,000 from a subunits leads to a loss of membrane localization (6, 7)
We have recently demonstrated that iloprosta, stable prostacyclin analogue, binds to the membrane preparedfrom mouse mastocyand inhibitionof adenylate cyclase, respectively; Go, a similar GTP- tomaP-815 cells, neoplastic mast cells, andenhancesthe binding proteinof unknown function; Gsae, xample of the nomencla- activity of adenylate cyclase in a GTP-dependentmanner ture used to designate a specific a subunit of the indicated G-protein; PGI, prostacyclin; GTPyS, guanosine 5‘-O-(thiotriphosphate);Gpp (NH)p, guanyl-5’-(@,y-imido)diphosphate;SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Hepes, 4-(2-bydroxyethy1)-1-piperazineethanesulfonicacid; GDPPS, guanyl-5’-yl thiophosphate

Summary

Introduction

Pcukm4yraD2ioct-neaslaaadt-lnspsedractoianh4mtctoe5itulbi-ienlvkoarsiDdatteyyradt,e,opacRxracaioMdutnitsev-e/enc1eidyan, tlwstafahriltienoyethmzrteehcttldhyeheeceaAlsmaeaDmsneeoPmetfGim-abbrsciobarbtatriohcavnasnie4yrtey2bl.aa-ontTaxidoanhyonndelaf-dttitterlheoer4en-l-5e--asteiehenofnaftvteesepacrcteosbhcureiafebcnynougutnaehpsiaetlsbsiunlf(emgu1.ne)aIcd.Fnntdritoocomnsaoeponrfptvtrehaeaaesapritsra,htryotatdihncr0eiuon-alyptaneshrrucoahGbibcdoui-tecfrnpniotirtyrtoisc,tteahtalhielnryeeafi,&unwsnuricsbtethuciuobendnpuiiattnfolfilroety-srnof Gsa from the membrane were markedly suppressedplasma membranes (3). Membrane even after activation withGTP-yS inbovine brain iloprost induced the translocationof both 42- and 45- andhumanneutrophilmembranes(4).ManyG-protein a kDa Gsa from the membrane to the cytosol, 45-kDa subunits aremyristoylated, the siteof modification being the

Results

Conclusion