TY - JOUR
T1 - Grab recruitment by Rab27A-Rabphilin3a triggers Rab3A activation in human sperm exocytosis
AU - Quevedo, María Florencia
AU - Bustos, Matías Alberto
AU - Masone, Diego
AU - Roggero, Carlos Marcelo
AU - Bustos, Diego Martín
AU - Tomes, Claudia Nora
N1 - Funding Information:
This work was supported by Agencia Nacional de Promoción Científica y Tecnológica (ANPCYT, grant numbers PICT2010-0342 and PICT-2013-1216 ) and by Secretaría de Ciencia y Técnica - Universidad Nacional de Cuyo (grant number 06/J484 ) to C.N.T; by MINCyT -Sistema Nacional de Computación de Alto Rendimiento (SNCAD), Proyectos Acelerados de Cálculo (IPAC, grant number SACT 017-00721036 ) to D.M.B and by MINCyT-SNCAD (grant number 2017-75-APN-SECACT#MCT ) to DM.
Publisher Copyright:
© 2019 Elsevier B.V.
PY - 2019/4
Y1 - 2019/4
N2 - Sperm must undergo the regulated exocytosis of its dense core granule (the acrosome reaction, AR) to fertilize the egg. We have previously described that Rabs3 and 27 are organized in a RabGEF cascade within the signaling pathway elicited by exocytosis stimuli in human sperm. Here, we report the identity and the role of two molecules that link these secretory Rabs in the RabGEF cascade: Rabphilin3a and GRAB. Like Rab3 and Rab27, GRAB and Rabphilin3a are present, localize to the acrosomal region and are required for calcium-triggered exocytosis in human sperm. Sequestration of either protein with specific antibodies introduced into streptolysin O-permeabilized sperm impairs the activation of Rab3 in the acrosomal region elicited by calcium, but not that of Rab27. Biochemical and functional assays indicate that Rabphilin3a behaves as a Rab27 effector during the AR and that GRAB exhibits GEF activity toward Rab3A. Recombinant, active Rab27A pulls down Rabphilin3a and GRAB from human sperm extracts. Conversely, immobilized Rabphilin3a recruits Rab27 and GRAB; the latter promotes Rab3A activation. The enzymatic activity of GRAB toward Rab3A was also suggested by in silico and in vitro assays with purified proteins. In summary, we describe here a signaling module where Rab27A-GTP interacts with Rabphilin3a, which in turn recruits a guanine nucleotide-exchange activity toward Rab3A. This is the first description of the interaction of Rabphilin3a with a GEF. Because the machinery that drives exocytosis is highly conserved, it is tempting to hypothesize that the RabGEF cascade unveiled here might be part of the molecular mechanisms that drive exocytosis in other secretory systems.
AB - Sperm must undergo the regulated exocytosis of its dense core granule (the acrosome reaction, AR) to fertilize the egg. We have previously described that Rabs3 and 27 are organized in a RabGEF cascade within the signaling pathway elicited by exocytosis stimuli in human sperm. Here, we report the identity and the role of two molecules that link these secretory Rabs in the RabGEF cascade: Rabphilin3a and GRAB. Like Rab3 and Rab27, GRAB and Rabphilin3a are present, localize to the acrosomal region and are required for calcium-triggered exocytosis in human sperm. Sequestration of either protein with specific antibodies introduced into streptolysin O-permeabilized sperm impairs the activation of Rab3 in the acrosomal region elicited by calcium, but not that of Rab27. Biochemical and functional assays indicate that Rabphilin3a behaves as a Rab27 effector during the AR and that GRAB exhibits GEF activity toward Rab3A. Recombinant, active Rab27A pulls down Rabphilin3a and GRAB from human sperm extracts. Conversely, immobilized Rabphilin3a recruits Rab27 and GRAB; the latter promotes Rab3A activation. The enzymatic activity of GRAB toward Rab3A was also suggested by in silico and in vitro assays with purified proteins. In summary, we describe here a signaling module where Rab27A-GTP interacts with Rabphilin3a, which in turn recruits a guanine nucleotide-exchange activity toward Rab3A. This is the first description of the interaction of Rabphilin3a with a GEF. Because the machinery that drives exocytosis is highly conserved, it is tempting to hypothesize that the RabGEF cascade unveiled here might be part of the molecular mechanisms that drive exocytosis in other secretory systems.
KW - Exocytosis
KW - GRAB
KW - RabGEF cascade
KW - Rabphilin3a
KW - Sperm
UR - http://www.scopus.com/inward/record.url?scp=85060041806&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85060041806&partnerID=8YFLogxK
U2 - 10.1016/j.bbamcr.2018.12.005
DO - 10.1016/j.bbamcr.2018.12.005
M3 - Article
C2 - 30599141
AN - SCOPUS:85060041806
SN - 0167-4889
VL - 1866
SP - 612
EP - 622
JO - Biochimica et Biophysica Acta - Molecular Cell Research
JF - Biochimica et Biophysica Acta - Molecular Cell Research
IS - 4
ER -