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

Abstract

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 GTPRab27-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 (␤-␥-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.In eukaryotic cells, transport between distinct organelles is performed through vesicle trafficking. The final step of vesicle docking/fusion with target membrane is mediated by transsoluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) 3 complex bridging a vesicle and its target membrane (1). One of the key regulators for the SNARE complex formation is Rab GTPase (2, 3). So far, more than 60 members of Rab GTPases are identified in mammals, and they play critical roles in the specific transport pathways (2-4). Like other GTPases, the activity of Rab is regulated by its GDP/GTP cycle. Rab proteins have GTP-bound active and GDP-bound inactive forms. The activation process is performed by GDP/ GTP exchange mediated by the GDP/GTP exchange factor. GTP-bound Rabs execute their function by interaction with effector proteins. Then, GTP-Rab is inactivated into GDP-Rab by GTP hydrolysis that is mediated by the intrinsic GTPase activity and its enhancer, GTPase-activating protein. Furthermore, the Rab family has a unique regulatory protein named Rab GDP dissociation inhibitor (RabGDI), which extracts GDP-Rab from membrane into cytosol by forming a 1:1 complex and inhibits GDP/GTP exchange (3). RabGDI accompanies Rabs in cytosol to the correct organelles, where they are reactivated by the function of GDI dissociation factor (5-8).Although elucidation of the regulatory mechanism of the GDP/GTP cycle is crucial for understanding the functional mechanism of Rab GTPases, it has not been extensively investigated so far. Small GTPases belonging to Ras and Rho families are predominantly present in the GDP-bound forms under resting conditions and are transiently activated into GTPbound forms upon stimulation (9), indicating that these GTPases function as "switches" that transduce...

Rab27a and Rab27b are involved in stimulation-dependent RANKL release from secretory lysosomes in osteoblastic cells

“…Previous reports showed that Rab27a and Rab27b are likely to exist in their activated form (GTP-bound form) at the basal condition. (45) Therefore, Rab27a and Rab27b may not be responsible for triggering RANKL release in response to the RANK-mediated activation of a still-unknown signaling pathway, but rather osteoblastic cells may use the already-activated machinery of the Rab27-effector complex in stimulation-dependent RANKL release. We also showed that Slp4-a, Slp5, and Munc13-4, which are known effector molecules for Rab27a and Rab27b, are involved in this pathway.…”

The interplay between the Rab27A effectors Slp4-a and MyRIP controls hormone-evoked Weibel-Palade body exocytosis

“…40 Slow Rab27A cycling could reflect the fast constitutive GDP-GTP exchange and slow GTP hydrolysis rates reported to hold Rab27A predominantly as GTP-Rab27A on SGs. 37 Unlike for Rab3s, conversion to GDP-Rab27A is not associated with loss from membranes. 37 Whether this arises because Rab27A is a poor substrate for GDI or because access to GDI is hindered by binding to effectors in the GDP-state (eg, Slp4-a, coronin-3) 41,42 remains to be established.…”

“…37 Unlike for Rab3s, conversion to GDP-Rab27A is not associated with loss from membranes. 37 Whether this arises because Rab27A is a poor substrate for GDI or because access to GDI is hindered by binding to effectors in the GDP-state (eg, Slp4-a, coronin-3) 41,42 remains to be established. The cycling of EGFP-Slp4-a on mRFP-Rab27A-expressing WPBs mirrored the very slow cycling of Rab27A itself, presumably because Slp4-a, unlike other Rabs, binds to both GTP and GDP-bound states of Rab27A.…”

“…However, if endogenous Rab3B were more abundant than Rab27A at the protein level, as seen at the transcript level, then competition between Rab3B and Rab27A for shared molecular components could account for Rab3B-positive Rab27A-negative WPBs. Indeed, a very low GTPase activity for Rab27A may lend an additional competitive advantage to Rab3B in accessing REP. 37,38 Direct evidence for competition between secretory Rabs in HUVECs came from observations that EGFP-Rab27A or mEGFP-Rab3s' expression depleted endogenous WPB-Rab3B-or Rab27A-IR, respectively (supplemental Figure 7A-B). The primary point of competition between these Rabs is probably membrane insertion because neither cytosolic EGFP nor YFP-Rab1A, which uses distinct GEFs (and GTPase activating proteins), 39 displaced Rab3B from WPBs (supplemental Figure 7C).…”

Association of Single Nucleotide Polymorphism in Chromosome 11 with Autism Spectrum Disorder

“…A known function of this gene is related to Rab27, which governs the regulated exocytosis in non-neuronal cells, such as lytic granule secretion in cytotoxic T cells, production of insulin in pancreatic β-cells, as well as the release of histamine-containing granules in mast cells. Rab27 is also responsible for melanosome transport along the actin cytoskeleton in melanocytes (Kondo et al, 2006). As EXPH5 does not appear to be involved in neurological processes, it is probably not of notable significance in ASD.…”