Constitutive GDP/GTP exchange and secretion-dependent GTP hydrolysis activity for Rab27 in platelets.
We have previously demonstrated that Rab27 regulates dense granule secretion in platelets. Here, we analyzed the activation status of Rab27 using the thin layer chromatography method analyzing nucleotides bound to immunoprecipitated Rab27 and the pull-down method quantifying Rab27 bound to the GTP-Rab27-binding domain (synaptotagmin-like protein (Slp)-homology domain) of its specific effector, Slac2-b. We found that Rab27 was predominantly present in the GTP-bound form in unstimulated platelets due to constitutive GDP/GTP exchange activity. The GTP-bound Rab27 level drastically decreased due to enhanced GTP hydrolysis activity upon granule secretion. In permeabilized platelets, increase of Ca(2+) concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (beta-gamma-imidoguanosine 5'-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca(2+)-induced secretion. These data suggested that GTP hydrolysis of Rab27 was not necessary for inducing the secretion. Taken together, Rab27 is maintained in the active status in unstimulated platelets, which could function to keep dense granules in a preparative status for secretion.
Kondo
Hirokazu
H
Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
Shirakawa
Ryutaro
R
Higashi
Tomohito
T
Kawato
Mitsunori
M
Fukuda
Mitsunori
M
Kita
Toru
T
Horiuchi
Hisanori
H
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry*; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry*; D006801:Humans; D006868:Hydrolysis; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D013997:Time Factors; D020691:rab GTP-Binding Proteins / Q000737:chemistry*; D000075745:rab27 GTP-Binding Proteins
D048868:Adaptor Proteins, Signal Transducing; C514892:EXPH5 protein, human; D009711:Nucleotides; D000075745:rab27 GTP-Binding Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins; D002118:Calcium