suREJ3, a Polycystin-1 Protein, Is Cleaved at the GPS Domain and Localizes to the Acrosomal Region of Sea Urchin Sperm

Abstract

The sea urchin sperm acrosome reaction (AR) is a prerequisite for sperm-egg fusion. This report identifies sea urchin sperm receptor for egg jelly-3 (suREJ3) as a new member of the polycystin-1 family (the protein mutated in autosomal dominant polycystic kidney disease). suREJ3 is a multidomain, 2,681-amino acid, heavily glycosylated orphan receptor with 11 putative transmembrane segments (TMS) that localize to the plasma membrane covering the sperm acrosomal vesicle. Like the latrophilins and other members of the secretin family of G-protein-coupled receptors, suREJ3 is cleaved at the consensus GPS (G-protein-coupled receptor proteolytic site) domain. Antibodies to the extracellular 1,455-residue NH 2 -terminal portion identify a band at 250 kDa that shifts in electrophoretic mobility to 180 kDa upon glycosidase digestion. Antibodies to the 1,226-residue COOH-terminal portion identify a band at 150 kDa that shifts to 140 kDa after glycosidase treatment. Antibodies to both portions of suREJ3 localize exclusively to the plasma membrane over the acrosomal vesicle. Immunoprecipitation shows that both portions of suREJ3 are associated in detergent extracts. This is the first report showing that a polycystin family member is cleaved at the GPS domain. Localization of suREJ3 to the acrosomal region provides the first suggestion for the role of a polycystin-1 protein (components of nonselective cation channels) in a specific cellular process.

A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets

“…The apparent asynchronous behavior of mammalian spermatozoa coupled with the paucity of female gametes (eggs) prompted many to use invertebrate animal models to understand the molecular basis of fertilization (29,30). Functional bioassays allowed identification of important signaling components in the invertebrate sperm cells, and the subsequent identification of homologous genes in the mammal seemed a strong approach for unraveling fertilization mechanisms in the mammal.…”

Vasculostatin, a proteolytic fragment of Brain Angiogenesis Inhibitor 1, is an antiangiogenic and antitumorigenic factor

“…We searched the predicted protein sequence of BAI1 using Simple Modular Architecture Research Tool (SMART) for consensus sequence of known proteolytic cleavage sites and identified a G-protein-coupled receptor proteolytic cleavage site (GPS) at the junction of the N-terminal extracellular region with the first transmembrane domain (Figure 1a). GPS domains are approximately 50 residues long containing either two or four conserved cysteinecs and are found by sequence homology in otherwise functionally unrelated receptors including latrophilin-1 (CL1), hCD97, Flamingo-1, sea urchin sperm receptor for egg jelly (suREJ3) and Ig Hepta (Gray et al, 1996;Krasnoperov et al, 2002;Mengerink et al, 2002). Alignment of the GPS domains in BAI1 and other GPS-containing receptors is shown in Figure 1c.…”

An ATP-binding Cassette Transporter Is a Major Glycoprotein of Sea Urchin Sperm Membranes

“…Peptide Sequencing and Sequence Analysis-Several high molecular weight proteins are enriched in WGA eluates of solubilized sperm protein when compared with the starting material, including suREJ1 and suREJ3 (6,7). The 220-kDa band is enriched in the eluate and can also be detected in the starting material (Fig.…”

“…Antibody Production-A portion of suABCA DNA corresponding to Ala 961 -Pro 1158 was ligated into the pET15b vector (Novagen), which contains an NH 2 -terminal His tag, and bacterially expressed according to a previously published protocol (7). The resulting purified recombinant protein, ABCe, was separated by SDS-PAGE and negatively stained with cupric chloride (20).…”

“…Wheat germ agglutinin (WGA) blocks the egg jelly-induced AR of sea urchin sperm (5). One WGA-binding protein, suREJ1, has been demonstrated to be involved in triggering the AR (6), and another, suREJ3, has been implicated in this process (7). This paper identifies another major WGA-binding protein of sea urchin sperm as an ATP-binding cassette (ABC) transporter, most closely related to human ABCA3.…”