Grab recruitment by Rab27A-Rabphilin3a triggers Rab3A activation in human sperm exocytosis

QUEVEDO, MARÍA FLORENCIA; BUSTOS, MATÍAS ALBERTO; MASONE, DIEGO; ROGGERO, CARLOS MARCELO; BUSTOS, DIEGO MARTÍN; TOMES, CLAUDIA NORA

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2019 vol. 1866 p. 612 - 622

0167-4889

Sperm must undergo the regulated exocytosis of its dense core granule (the acrosome reaction, AR) to fertilizethe egg. We have previously described that Rabs3 and 27 are organized in a RabGEF cascade within the signalingpathway elicited by exocytosis stimuli in human sperm. Here, we report the identity and the role of two mo-lecules 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-triggeredexocytosis in human sperm. Sequestration of either protein with specific antibodies introduced into streptolysinO-permeabilized sperm impairs the activation of Rab3 in the acrosomal region elicited by calcium, but not thatof Rab27. Biochemical and functional assays indicate that Rabphilin3a behaves as a Rab27 effector during theAR and that GRAB exhibits GEF activity toward Rab3A. Recombinant, active Rab27A pulls down Rabphilin3aand GRAB from human sperm extracts. Conversely, immobilized Rabphilin3a recruits Rab27 and GRAB; thelatter promotes Rab3A activation. The enzymatic activity of GRAB toward Rab3A was also suggested by in silicoand in vitro assays with purified proteins. In summary, we describe here a signaling module where Rab27A-GTPinteracts with Rabphilin3a, which in turn recruits a guanine nucleotide-exchange activity toward Rab3A. This isthe first description of the interaction of Rabphilin3a with a GEF. Because the machinery that drives exocytosis ishighly conserved, it is tempting to hypothesize that the RabGEF cascade unveiled here might be part of themolecular mechanisms that drive exocytosis in other secretory systems