Conserved sequences of sperm-activating peptide and its receptor throughout evolution, despite speciation in the sea star Asterias amurensis and closely related species

The asteroidal sperm-activating peptides (asterosaps) from the egg jelly bind to their sperm receptor, a membrane-bound guanylate cyclase, on the tail to activate sperm in sea stars. Asterosaps are produced as single peptides and then cleaved into shorter peptides. Sperm activation is followed by the acrosome reaction, which is subfamily specific. In order to investigate the molecular details of the asterosap-receptor interaction, corresponding cDNAs have been cloned, sequenced and analysed from the Asteriinae subfamily including Asterias amurensis, A. rubens, A. forbesi and Aphelasterias japonica, as well as Distolasterias nipon from the Coscinasteriinae subfamily. Averages of 29% and 86% identity were found from the deduced amino acid sequences in asterosap and its receptor extracellular domains, respectively, across all species examined. The phylogenic tree topology for asterosap and its receptor was similar to that of the mitochondrial cytochrome c oxidase subunit I. In spite of a certain homology, the amino acid sequences exhibited speciation. Conservation was found in the asterosap residues involved in disulphide bonding and proteinase-cleaving sites. Conversely, similarities were detected between potential asterosap-binding sites and the structure of the atrial natriuretic peptide receptor. Although the sperm-activating peptide and its receptor share certain common sequences, they may serve as barriers that ensure speciation in the sea star A. amurensis and closely related species.     

Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella

Adenylyl cyclases (ACs) synthesize cAMP and are present in cells as transmembrane AC and soluble AC (sAC). In sperm, the cAMP produced regulates ion channels and it also activates protein kinase-A that in turn phosphorylates specific axonemal proteins to activate flagellar motility. In mammalian sperm, sAC localizes to the midpiece of flagella, whereas in sea urchin sperm sAC is along the entire flagellum. Here we show that in sea urchin sperm, sAC is complexed with proteins of the plasma membrane and axoneme. Immunoprecipitation shows that a minimum of 10 proteins is tightly associated with sAC. Mass spectrometry of peptides derived from these proteins shows them to be: axonemal dynein heavy chains 7 and 9, sperm specific Na+/H+ exchanger, cyclic nucleotide-gated ion channel, sperm specific creatine kinase, membrane bound guanylyl cyclase, cyclic GMP specific phosphodiesterase 5A, the receptor for the egg peptide speract, and alpha- and beta-tubulins. The sAC-associated proteins could be important in linking membrane signal transduction to energy utilisation in the regulation of flagellar motility.     

Identification of a cell surface receptor common to germ and somatic cells.

The plasma membrane forms of guanylyl cyclase constitute a diverse family of cell surface receptors. An mRNA for the enzyme/receptor was first cloned from sea urchin testis after cross-linking studies suggested that guanylyl cyclase was a sperm receptor for egg peptides. The enzyme/receptor was shown to contain a single putative transmembrane domain, a large extracellular region that presumably binds peptide ligands, and an intracellular region that contains a protein kinase-like and a cyclase catalytic domain. The sea urchin cDNA was then used to isolate positive-hybridizing clones from mammalian tissues. At least two forms recognize natriuretic peptides and one form recognizes the heat-stable enterotoxins. In the case of the enterotoxin receptor, it remains to be shown whether or not an endogenous ligand exists that regulates enzyme activity. The discovery of this cell surface receptor family presents a new paradigm for second messenger signalling in that a low-molecular weight second messenger (cyclic GMP) is produced by the same protein that binds the extracellular ligand.     

Receptor-mediated regulation of guanylate cyclase activity in spermatozoa

Two peptides, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH2), which activate sperm respiration and motility and elevate cyclic GMP concentrations in a species-specific manner, were tested for effects on guanylate cyclase activity. The guanylate cyclase of sea urchin spermatozoa is a glycoprotein and it is localized entirely on the plasma membrane. When intact sea urchin sperm cells were incubated with the appropriate peptide for time periods as short as 5 s and subsequently homogenized in detergent, guanylate cyclase activity was found to be as low as 10% of the activity of cells not treated with peptide. The peptides showed complete species specificity and analogues of one peptide (speract) caused decreases in enzyme activity coincident with their receptor binding properties. The peptides did not inhibit enzyme activity when added after detergent solubilization of the enzyme. When detergent-solubilized spermatozoa were incubated at 22 degrees C, guanylate cyclase activity declined in previously nontreated cells to the peptide-treated level. The rate of decline was dependent on temperature and protein concentration. When spermatozoa were first incubated with 32P, the decrease in guanylate cyclase activity was accompanied by a shift in the apparent molecular weight of a major plasma membrane protein (160,000-150,000) and a loss of 32P label from the 160,000 band. Other agents (Monensin A, NH4Cl) which were capable of stimulating sperm respiration and motility also caused decreases of guanylate cyclase activity when added to intact but not detergent-solubilized spermatozoa. The maximal decrease in guanylate cyclase activity occurred 5-10 min after addition of these agents. The enzyme response to Monensin A required extracellular Na+ suggestive that the ionophore caused the effect on guanylate cyclase activity by virtue of its ability to catalyze Na+/H+ exchange. These studies demonstrate that guanylate cyclase activity of sperm cells can be altered by the specific interaction of egg-associated peptides with their plasma membrane receptors.     

FMRFamide related peptide ligands activate the Caenorhabditis elegans orphan GPCR Y59H11AL.1

G-protein coupled receptors (GPCRs) are ancient molecules that can sense environmental and physiological signals. Currently, the majority of the predicted Caenorhabditis elegans GPCRs are orphan. Here, we describe the characterization of such an orphan C. elegans GPCR, which is categorized in the tachykinin-like group of receptors. Since the C. elegans genome predicts only one tachykinin-like peptide (SFDRMGGTEFGLM), which could not activate the receptor, we hypothesized that one or some of the numerous FMRFamide related peptides (FaRPs) could be the cognate ligands for this receptor. This hypothesis was based on the suggestion that RFamides may be ancestral neuropeptides, from which a lot of the amidated neuropeptides, including tachykinins, derived. Indeed, we found that the orphan receptor encoded by the Y59H11AL.1 gene is activated by several C. elegans neuropeptides, including SPMERSAMVRFamide. These peptides activate the receptor in a concentration-dependent way.     

Fate of the sea urchin egg receptor for sperm following fertilization

The sea urchin egg receptor for sperm is a 350 kDa glycoprotein containing a large extracellular domain that contains the sperm binding site, a transmembrane domain and a short COOH- terminal intracellular domain. During oogenesis, the receptor protein is first detected in Golgi-associated vesicles and cortical granules. Not until the egg is mature does the receptor appear on the cell surface; at this stage the intact receptor is found in approximately equal quantities on the egg cell surface and in cortical granules. As a potentially unique type of receptor, we were interested in its fate following fertilization. Several techniques have revealed that, following sperm binding, the amount of receptor markedly decreases. Using western blot analysis as well as direct measurement of the receptor protein, it was found that the membrane-bound form of the receptor rapidly disappeared following sperm binding to the egg, with only 3% of the receptor remaining after 30 s. Analysis by immupoelectron microscopy revealed that 30 s after sperm binding, 30% of the initial level of receptor was present. This remaining 30% was found mostly within the perivitelline space formed by the raised fertilization envelope. The disparity between these two sets of results (i.e. 3 vs 30%) is most likely accounted for by the exocytosis of receptor molecules from cortical granules; this fraction of the receptor would have been lost during isolation of the membrane-bound form of the receptor. Thus, unlike other cell surface receptors, the sea urchin egg receptor for sperm is not endocytosed and recycled following ligand binding. Rather, it disappears, presumably as a result of proteolysis. Transiently, the cortical granule form of the receptor is found released into the perivitelline space where it may bind to sperm and thereby prevent polyspermy. Despite the apparent secretion of this form of the receptor, experiments with antibodies to the extracellular and intracellular domains indicate that the receptors in cortical granules and in the plasmic membrane are similar, if not identical.     

Characterization of the sperm receptor on the surface of eggs of Strongylocentrotus purpuratus

Eggs of the sea urchins Strongylocentrotus purpuratus and Arbacia punctulata bind sperm with a high degree of species specificity. By use of an in vitro assay that utilizes bindin (the protein from sperm that mediates sperm-egg binding) egg surface-derived glycoconjugates that function as receptors in this adhesion process have been identified and purified. These glycoconjugates are of extraordinarily high molecular weight and exhibit some properties expected for a proteoglycan. The isolated receptors from both species bind to sperm and inhibit fertilization species specifically. Both receptors contain active carbohydrate-rich fragments that can be liberated by proteolytic digestion. The carbohydrate-rich receptor fragment from S. purpuratus is a very high-molecular-weight (>10⁶), negatively charged glycosaminoglycan-like polymer containing fucose, galactosamine, iduronic acid, and sulfate esters. By contrast, the carbohydrate-rich fragment derived from the A. punctulata receptor is of defined molecular weight (6000) and has no net charge. Incubation of acrosome-reacted sperm with nanomolar amounts of the carbohydrate-rich fragments from either species results in inhibition of fertilization, indicating that these receptor fragments retain sperm binding activity. However, studies utilizing heterologous gametes show that the carbohydrate-rich receptor fragments are not species specific in binding. Thus, it appears that although the carbohydrate chains of the receptor are an adhesive element of the receptor, the intact glycoconjugate is required for species-specific binding.     

A tale of two cells: endocannabinoid-signaling regulates functions of neurons and sperm

Sea urchin and human sperm contain receptors for neurotransmitters and psychoactive drugs, including cannabinoid receptors (CNRs). Anandamide, arachidonoylethanolamide (AEA), is a lipid-signal molecule that is an endogenous agonist for CNRs. AEA is enyzmatically released from membrane phospholipids when neurons are stimulated. Retrograde AEA signals from depolarized postsynaptic neurons inhibit neurotransmitter release at synapses in mammalian brain. Analogous processes regulate sperm functions during fertilization in sea urchins. AEA and (-)delta9tetrahydrocannabinol [(-)delta9THC], the major psychoactive constituent of marijuana, inhibit fertilization by blocking acrosomal exocytosis/acrosome reactions (AR) stimulated by egg jelly. The acrosome is a Golgi-derived secretory granule in sperm analogous to synaptic vesicles in neurons. AEA and (-)delta9THC do not block ionophore-induced AR, suggesting that they inhibit AR by modulating signal transduction event(s) before opening of ion channels. Unfertilized sea urchin eggs have enzymes required to release AEA from membrane phospholipids. These results indicate that sea urchin eggs may release AEA after activation by the fertilizing sperm. Released AEA may then react with CNRs in nearby sperm to block AR, thereby helping to prevent polyspermy. AEA is present in human seminal plasma, midcycle oviductal fluid, and follicular fluid. Sperm are sequentially exposed to these fluids as they move from the vagina to the site of fertilization in the oviduct. R-methanandamide (AM-356), a metabolically stable AEA analog, and (-)delta9THC modulate capacitation and fertilizing potential of human sperm in vitro. These findings suggest that AEA signaling directly affects sperm functions required for fertilization and provide additional evidence for common signaling processes in neurons and sperm.     

Synchronous modulation of cell surface lectin and its receptor in a homologous cell population: a novel mechanism of cellular regulation

Testicular immotile sperm undergo maturation during epididymal transit when these cells pass through caput, corpus, and cauda-epididymal regions. Maturing goat spermatozoa specifically at the distal corpus epididymal stage show head-to-head autoagglutination when incubated in vitro in a modified Ringer's solution. Here, we show the biochemical mechanism of autoagglutination event and its functional significance. A lectin-like molecule located on sperm surface specifically interacts with its receptor of the neighboring homologous cells to cause autoagglutination. Lectin is a Ca++-dependent galactose-specific protein. Failure of the pre- and post-distal corpus sperm to show autoagglutination is due to lack of lectin-like molecule and its receptors, respectively. Maturing sperm at distal corpus stage acquire lectin-like molecule followed by sharp disappearance of its receptor, and this event is synchronously associated with the initiation of sperm forward motility that is essential for fertilization in vivo. Lectin and its receptor isolated from sperm plasma membrane showed high efficacy for blocking autoagglutination phenomenon. The data are consistent with the view that synchronous modulation of homologous cell surface lectin and their receptors constitutes a novel mechanism for cellular regulation by generating waves of signals by manipulating lectin-sugar-dependent "self-talk" and cell-cell "cross-talk".     

Bitter taste receptor T2R1 is activated by dipeptides and tripeptides

Bitter taste signaling in humans is mediated by a group of 25 bitter receptors (T2Rs) that belong to the G-protein coupled receptor (GPCR) family. Previously, several bitter peptides were isolated and characterized from bitter tasting food protein derived extracts, such as pea protein and soya bean extracts. However, the molecular targets or receptors in humans for these bitter peptides were poorly characterized and least understood. In this study, we tested the ability of the bitter tasting tri- and di-peptides to activate the human bitter receptor, T2R1. In addition, we tested the ability of peptide inhibitors of the blood pressure regulatory protein, angiotensin converting enzyme (ACE) to activate T2R1. Using a heterologous expression system, T2R1 gene was transiently expressed in C6-glioma cells and changes in intracellular calcium was measured following addition of the peptides. We found that the bitter tasting tri-peptides are more potent in activating T2R1 than the di-peptides tested. Among the peptides examined, the bitter tri-peptide Phe-Phe-Phe (FFF), is the most potent in activating T2R1 with an EC₅₀ value in the micromolar range. Furthermore, to elucidate the potential ligand binding pocket of T2R1 we used homology molecular modeling. The molecular models showed that the bitter peptides bind within the same binding pocket on the receptor. The ligand binding pocket in T2R1 is present on the extracellular surface of the receptor, and is formed by the transmembrane helices 1, 2, 3 and 7 and with extracellular loops 1 and 2 forming a cap like structure on the binding pocket.     

Derivatives of speract are associated with the eggs of Lytechinus pictus sea urchins

Two peptides associated with the eggs of the sea urchin, Lytechinus pictus, which stimulate L. pictus but not Arbacia punctulata sperm respiration rates, were purified and their amino acid sequences determined. The peptides (Gly-Phe-Asp-Leu-Thr-Gly-Gly-Gly-Val-Gln and Phe-Asp-Leu-Thr-Gly-Gly-Gly-Val-Gln) were found to be structurally similar to the peptide, speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly). Chemical synthesis of the two peptides confirmed their ability to activate sperm respiration. The peptides had equivalent biological activity with half-maximal stimulation of respiration rates and of cyclic nucleotide concentrations occurring at 60 pM and 700 pM, respectively. Addition of the peptides to intact spermatozoa resulted in the rapid appearance of a newly-stained protein on Na X dodecyl X SOl polyacrylamide gels (Mr = 140,000); one-half maximal formation of the Mr 140,000 protein occurred at about 20-100 nM peptide.     

Receptors that mediate cellular dependence

Cells depend for their survival on stimulation by trophic factors and other prosurvival signals, the withdrawal of which induces apoptosis, both via the loss of antiapoptotic signaling and the activation of proapoptotic signaling via specific receptors. These receptors, dubbed dependence receptors, activate apoptotic pathways following the withdrawal of trophic factors and other supportive stimuli. Such receptors may feature in developmental cell death, carcinogenesis (including metastasis), neurodegeneration, and possibly subapoptotic events such as neurite retraction and somal atrophy. Mechanistic studies of dependence receptors suggest that these receptors form ligand-dependent complexes that include specific caspases. Complex formation in the absence of ligand leads to caspase activation by a mechanism that is typically dependent on caspase cleavage of the receptor itself, releasing proapoptotic peptides. Cellular dependence receptors, considered in the aggregate, may thus form a system of molecular integration, analogous to the electrical integration system provided by dendritic arbors in the nervous system.     

A mechanism for SRC kinase-dependent signaling by noncatalytic receptors

A fundamental issue in cell biology is how signals are transmitted across membranes. A variety of transmembrane receptors, including multichain immune recognition receptors, lack catalytic activity and require Src family kinases (SFKs) for signal transduction. However, many receptors only bind and activate SFKs after ligand-induced receptor dimerization. This presents a conundrum: How do SFKs sense the dimerization of receptors to which they are not already bound? Most proposals for resolving this enigma invoke additional players, such as lipid rafts or receptor conformational changes. Here we used simple thermodynamics to show that SFK activation is a natural outcome of clustering of receptors with SFK phosphorylation sites, provided that there is phosphorylation-dependent receptor-SFK association and an SFK bound to one receptor can phosphorylate the second receptor or its associated SFK in a dimer. A simple system of receptor, SFK, and an unregulated protein tyrosine phosphatase (PTP) can account for ligand-induced changes in phosphorylation observed in cells. We suggest that a core signaling system comprising a receptor with SFK phosphorylation sites, an SFK, and an unregulated PTP provides a robust mechanism for transmembrane signal transduction. Other events that regulate signaling in specific cases may have evolved for fine-tuning of this basic mechanism.     

Targeting Eph receptors with peptides and small molecules: progress and challenges

The Eph receptors are a large family of receptor tyrosine kinases. Their kinase activity and downstream signaling ability are stimulated by the binding of cell surface-associated ligands, the ephrins. The ensuing signals are bidirectional because the ephrins can also transduce signals (known as reverse signals) following their interaction with Eph receptors. The ephrin-binding pocket in the extracellular N-terminal domain of the Eph receptors and the ATP-binding pocket in the intracellular kinase domain represent potential binding sites for peptides and small molecules. Indeed, a number of peptides and chemical compounds that target Eph receptors and inhibit ephrin binding or kinase activity have been identified. These molecules show promise as probes to study Eph receptor/ephrin biology, as lead compounds for drug development, and as targeting agents to deliver drugs or imaging agents to tumors. Current challenges are to find (1) small molecules that inhibit Eph receptor-ephrin interactions with high binding affinity and good lead-like properties and (2) selective kinase inhibitors that preferentially target the Eph receptor family or subsets of Eph receptors. Strategies that could also be explored include targeting additional Eph receptor interfaces and the ephrin ligands.     

The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways.

Bone morphogenetic proteins (BMPs) are multifunctional proteins regulating cell growth, differentiation, and apoptosis. BMP-2 signals via two types of receptors (BRI and BRII) that are expressed at the cell surface as homomeric as well as heteromeric complexes. Prior to ligand binding, a low but measurable level of BMP-receptors is found in preformed hetero-oligomeric complexes. The major fraction of the receptors is recruited into hetero-oligomeric complexes only after ligand addition. For this, BMP-2 binds first to the high affinity receptor BRI and then recruits BRII into the signaling complex. However, ligand binding to the preformed complex composed of BRII and BRI is still required for signaling, suggesting that it may mediate activating conformational changes. Using several approaches we have addressed the following questions: (i) Are preformed complexes incompetent of signaling in the absence of BMP-2? (ii) Which domains of the BRII receptors are essential for this complex formation? (iii) Are there differences in signals sent from BMP-induced versus preformed receptor complexes? By measuring the activation of Smads, of p38 MAPK and of alkaline phosphatase, we show that the ability of kinase-deficient BRII receptor mutants to inhibit BMP signaling depends on their ability to form heteromeric complexes with BRI. Importantly, a BRII mutant that is incapable in forming preassembled receptor complexes but recruits into a BMP-induced receptor complex does not interfere with the Smad pathway but does inhibit the induction of alkaline phosphatase as well as p38 phosphorylation. These results indicate that signals induced by binding of BMP-2 to preformed receptor complexes activate the Smad pathway, whereas BMP-2-induced recruitment of receptors activates a different, Smad-independent pathway resulting in the induction of alkaline phosphatase activity via p38 MAPK.