Monoclonal antibodies increase intracellular Ca2+ in sea urchin spermatozoa.

Trimmer, James S.; Schackmann, Robert W.; Vacquier, Victor D.

doi:10.1073/pnas.83.23.9055

Cited by 67 publications

(41 citation statements)

References 36 publications

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“…It is possible that the cleavage of polycystin-1 at the GPS leads to a conformational change of the TM and the cytoplasmic domains, which in turn influences the ability of polycystin-1 to co-assemble with polycystin-2 to form a calcium-permeable nonselective ion channel. On this note, it is interesting that both latrophilin/CL-1 and suREJ1 have also been shown to support Ca 2ϩ influx, although it is not known whether the GPS cleavage is required for this function (57,58). We have recently shown that both EMR2 and CD97 can act as an adhesion molecule, capable of binding to the cognate ligands on the cell surface (44,48).…”

Section: Fig 6 Emr2 Autoproteolysis Is An Intramolecular Reactionmentioning

confidence: 99%

Autocatalytic Cleavage of the EMR2 Receptor Occurs at a Conserved G Protein-coupled Receptor Proteolytic Site Motif

Lin¹,

Chang²,

Davies³

et al. 2004

Journal of Biological Chemistry

192

187

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Post-translational cleavage at the G protein-coupled receptor proteolytic site (GPS) has been demonstrated in many class B2 G protein-coupled receptors as well as other cell surface proteins such as polycystin-1. However, the mechanism of the GPS proteolysis has never been elucidated. Here we have characterized the cleavage of the human EMR2 receptor and identified the molecular mechanism of the proteolytic process at the GPS. Proteolysis at the highly conserved His-Leu2Ser 518 cleavage site can occur inside the endoplasmic reticulum compartment, resulting in two protein subunits that associate noncovalently as a heterodimer. Site-directed mutagenesis of the P ؉1 cleavage site (Ser 518) shows an absolute requirement of a Ser, Thr, or Cys residue for efficient proteolysis. Substitution of the P ؊2 His residue to other amino acids produces slow processing precursor proteins, which spontaneously hydrolyze in a defined cellfree system. Further biochemical characterization indicates that the GPS proteolysis is mediated by an autocatalytic intramolecular reaction similar to that employed by the N-terminal nucleophile hydrolases, which are known to activate themselves by self-catalyzed cisproteolysis. We propose here that the autoproteolytic cleavage of EMR2 represents a paradigm for the other GPS motif-containing proteins and suggest that these GPS proteins belong to a cell surface receptor subfamily of N-terminal nucleophile hydrolases.

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Section: Fig 6 Emr2 Autoproteolysis Is An Intramolecular Reactionmentioning

confidence: 99%

Autocatalytic Cleavage of the EMR2 Receptor Occurs at a Conserved G Protein-coupled Receptor Proteolytic Site Motif

Lin¹,

Chang²,

Davies³

et al. 2004

Journal of Biological Chemistry

192

187

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“…REJ1 has only one transmembrane segment at its extreme COOH terminus with only 15 residues being putatively cytoplasmic; therefore, REJ1 cannot be a pore-forming ion channel subunit. Because some (but not all) monoclonal antibodies to REJ1 induce Ca 2ϩ elevations and the AR, REJ1 must be a regulator of sperm Ca 2ϩ channels (12,13). The location of REJ1 over the acrosomal vesicle supports its role in AR regulation.…”

Section: Discussionmentioning

confidence: 99%

“…An affinity column of REJ1 binds only FSP when crude EJ is applied (6). Monoclonal antibodies to REJ1 induce Ca 2ϩ influx into sperm (13) and induce the AR (14). Approximately 1000 residues of REJ1 consist of a domain named "the REJ module," which is found in only one other protein family, the polycystin-1s (PKD1; Refs.…”

mentioning

confidence: 99%

High Molecular Mass Egg Fucose Sulfate Polymer Is Required for Opening Both Ca2+ Channels Involved in Triggering the Sea Urchin Sperm Acrosome Reaction

Hirohashi

Vacquier

2002

Journal of Biological Chemistry

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A linear fucose sulfate polymer (FSP), >106 daltons, is a major component of sea urchin egg jelly. FSP induces the sperm acrosome reaction (AR), an exocytotic process required for animal fertilization. Two Ca 2؉ channels activate during AR induction, the first opens 1 s after FSP addition, and the second opens 5 s after the first. Mild acid hydrolysis of FSP results in a linear decrease in polymer size. The ability of FSP to induce the AR and activate sperm Ca 2؉ channels decreases with increasing time of hydrolysis. Hydrolyzed FSP of ϳ60 kDa blocks intact FSP from inducing the AR. At 44 g/ml hydrolyzed FSP, Ca 2؉ entry into sperm is almost equal to that occurring in 3.8 g/ml intact FSP; however the AR is not induced. The sperm acrosome reaction (AR) 1 is required for animal fertilization and is a potential target for the development of novel methods of non-hormonal contraception. Sea urchin spermatozoa are ideal for studying signal transduction underlying the animal sperm AR because they can be obtained as pure cells in vast quantities at low cost. The AR is triggered when sperm contact the jelly layer surrounding the egg (EJ). Morphologically, the AR involves the exocytosis of the acrosomal vesicle and the polymerization of actin to form the acrosomal process; both events are required for sperm to bind to and fuse with eggs. Physiologically, the AR requires the influx of Ca 2ϩ and Na ϩ and the efflux of H ϩ and K ϩ ions (1, 2). There are two plasma membrane Ca 2ϩ channels involved in AR induction: the first is receptor-operated and opens 1 s after sperm contact EJ, the second opens 5 s after the first in response to increased intracellular pH (pH i ). The second channel can also transport Mn 2ϩ (2-4). The Ca 2ϩ channel blocker nisoldipine does not block Mn 2ϩ movement through the second channel but does block the first channel and hence also blocks the AR (3). Eighty percent of the mass of sea urchin EJ is a fucose sulfate polymer (FSP) of Ͼ1 million daltons (5). Purified FSP, having no amino acid content, induces the AR (5, 6). However, oligosaccharides of EJ glycoproteins substantially potentiate the FSP-induced AR, suggesting there is more than one receptor system regulating ion channels that trigger the AR (7). 2FSP is a linear polymer of ␣-L-1,3-fucose with a species-specific pattern of sulfation of the fucosyl residues (8). The sulfation pattern is responsible for FSP's species-specific induction of the AR (9, 10). FSP is also a potent inhibitor of human blood coagulation through its high affinity binding to heparin cofactor II (11).Receptor for egg jelly-1 (REJ1) is a 1450-amino acid glycoprotein located in the plasma membrane over the sea urchin sperm acrosomal vesicle and also on the sperm flagellum. Available data support the hypothesis that REJ1 is at least one of the sperm receptors for FSP (6, 12). Purified REJ1 neutralizes the AR activity of EJ (12). An affinity column of REJ1 binds only FSP when crude EJ is applied (6). Monoclonal antibodies to REJ1 induce Ca 2ϩ influx into sperm (13) and induce...

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“…Once again, systems in which in vitro fertilization was possible proved indispensable. Studies in algae and in sea urchins using known pharmacological inhibitors and monoclonal antibodies showed the importance of calcium ions in the process of egg activation and fertilization (Snell et al 1982, Trimmer et al 1986). …”

Section: Early Approachesmentioning

confidence: 99%

Molecular genetic approaches to studying fertilization in model systems

Geldziler¹,

Kadandale²,

Singson

2004

Reproduction

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In a wide range of experimental systems, a variety of both forward and reverse genetic approaches are becoming available for the study of the molecules involved in fertilization. An integration of these methods with the antibody-based and biochemical studies traditionally used in fertilization research is enabling rapid advancements in our understanding of this process. We highlight some of the recent advances resulting from these genetic methods and their applications in these systems.

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Monoclonal antibodies increase intracellular Ca2+ in sea urchin spermatozoa.

Cited by 67 publications

References 36 publications

Autocatalytic Cleavage of the EMR2 Receptor Occurs at a Conserved G Protein-coupled Receptor Proteolytic Site Motif

Autocatalytic Cleavage of the EMR2 Receptor Occurs at a Conserved G Protein-coupled Receptor Proteolytic Site Motif

High Molecular Mass Egg Fucose Sulfate Polymer Is Required for Opening Both Ca2+ Channels Involved in Triggering the Sea Urchin Sperm Acrosome Reaction

Molecular genetic approaches to studying fertilization in model systems

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