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J. Biol. Chem., Vol. 281, Issue 39, 28657-28665, September 29, 2006

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Constitutive GDP/GTP Exchange and Secretion-dependent GTP Hydrolysis Activity for Rab27 in Platelets^*

Hirokazu Kondo, Ryutaro Shirakawa¹, Tomohito Higashi¹, Mitsunori Kawato, Mitsunori Fukuda^¶, Toru Kita, and Hisanori Horiuchi²

From the Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, the Fukuda Initiative Research Unit, RIKEN, Wako, Saitama 351-0198, and the ^¶Laboratory of Membrane Trafficking Mechanisms, Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan

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 Ca²⁺ concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (--imidoguanosine 5'-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca²⁺-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.

Received for publication, April 5, 2006 , and in revised form, June 19, 2006.

^* This work was supported by Ministry of Education, Culture, Sports, Science, and Technology Research Grants, Japan, and by Health and Labor Sciences Research Grant for Cardiovascular Research from the Ministry of Health Labor and Welfare, Japan. This study was also supported in part by grants from the Takeda Science Foundation, Japan Heart Foundation Young Investigator's Research Grant and Japan Heart Foundation Pfizer Japan Inc. Grant for Research on Cardiovascular Disease. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipients of the Japan Society for the Promotion of Science Research Fellowship for Young Scientists.

² To whom correspondence should be addressed. Tel.: 81-75-751-3778; Fax: 81-75-751-3203; E-mail: horiuchi{at}kuhp.kyoto-u.ac.jp.

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