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The events that occur during chemotaxis of sperm are only partly known. As an essential step toward determining the underlying mechanism, we have recorded Ca2+ dynamics in swimming sperm of marine invertebrates. Stimulation of the sea urchin Arbacia punctulata by the chemoattractant or by intracellular cGMP evokes Ca2+ spikes in the flagellum. A Ca2+ spike elicits a turn in the trajectory followed by a period of straight swimming (‘turn-and-run’). The train of Ca2+ spikes gives rise to repetitive loop-like movements. When sperm swim in a concentration gradient of the attractant, the Ca2+ spikes and the stimulus function are synchronized, suggesting that precise timing of Ca2+ spikes controls navigation. We identified the peptide asterosap as a chemotactic factor of the starfish Asterias amurensis. The Ca2+ spikes and swimming behavior of sperm from starfish and sea urchin are similar, implying that the signaling pathway of chemotaxis has been conserved for almost 500 million years. [ABSTRACT FROM AUTHOR]

This study documents the identity of a calcium‐ regulated membrane guanylate cyclase transduction system in the photoreceptor‐bipolar synaptic region. The guanylate cyclase is the previously characterized ROS‐GC1 from the rod outer segments and its modulator is S100β. S100β senses increments in free Ca2+ and stimulates the cyclase. Specificity of photoreceptor guanylate cyclase activation by S100β is validated by the identification of two S100β‐regulatory sites. A combination of peptide competition, surface plasmon resonance binding and deletion mutation studies has been used to show that these sites are specific for S100β and not for another regulator of ROS‐GC1, guanylate cyclase‐activating protein 1. One site comprises amino acids (aa) Gly962–Asn981, the other, aa Ile1030–Gln1041. The former represents the binding site. The latter binds S100β only marginally, yet it is critical for control of maximal cyclase activity. The findings provide evidence for a new cyclic GMP transduction system in synaptic layers and thereby extend existing concepts of how a membrane‐bound guanylate cyclase is regulated by small Ca2+‐sensor proteins. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM) controls the activity of the rod cGMP-gated ion channel by decreasing the apparent cGMP affinity. We have examined the mechanism of this modulation using electrophysiological and biochemical techniques. Heteromeric channels, consisting of α- and β-subunits, display a high CaM sensitivity (EC50 ≤ 5 nM) similar to the native channel. Using surface plasmon resonance spectroscopy, we identified two unconventional CaM-binding sites (CaM1 and CaM2), one in each of the N- and the C-terminal regions of the β-subunit. Ca2+ co-operatively stimulates binding of CaM to these sites exactly within the range of [Ca2+] occurring during a light response. Deletion of the N-terminal CaM1 site results in channels that are no longer CaM-sensitive, whereas deletion of CaM2 has only minor effects. We discuss different models to explain the high-affinity binding of CaM. [ABSTRACT FROM AUTHOR]

G-protein βγ-subunits (Gβγ) are active transmembrane signalling components. Their function recently has been observed to be regulated by the cytosolic protein phosducin. We show here that a small fragment (amino acids 215–232) contained in the C-terminus of phosducin is sufficient for high-affinity interactions withGβγ. Corresponding peptides not only disrupt Gβγ-Gα interactions, as defined by Gβγ-stimulated GTPase activity of αo, but also other Gβγ-mediated functions. The NMR structure of a peptide encompassing this region shows a loop exposing the side chains of Glu223 and Tyr224, and peptides with a substitution of either of these amino acids show a complete loss of activity towardsGo. Mutation of this Tyr224 to Ala in full-length phosducin reduced the functional activity of phosducin to that of phosducin's concenisolated N-terminus, indicating the importance of this activaresidue within the short, structurally defined C-terminal segment. This small peptide derived from phosducin may represent a model of a Gβγ inhibitor, and illustrates the potential of small compounds to affectGβγ functions. [ABSTRACT FROM AUTHOR]

The events that occur during chemotaxis of sperm are only partly known. As an essential step toward determining the underlying mechanism, we have recorded Ca2+ dynamics in swimming sperm of marine invertebrates. Stimulation of the sea urchin Arbacia punctulata by the chemoattractant or by intracellular cGMP evokes Ca2+ spikes in the flagellum. A Ca2+ spike elicits a turn in the trajectory followed by a period of straight swimming ('turn-and-run'). The train of Ca2+ spikes gives rise to repetitive loop-like movements. When sperm swim in a concentration gradient of the attractant, the Ca2+ spikes and the stimulus function are synchronized, suggesting that precise timing of Ca2+ spikes controls navigation. We identified the peptide asterosap as a chemotactic factor of the starfish Asterias amurensis. The Ca2+ spikes and swimming behavior of sperm from starfish and sea urchin are similar, implying that the signaling pathway of chemotaxis has been conserved for almost 500 million years.

This study documents the identity of a calcium- regulated membrane guanylate cyclase transduction system in the photoreceptor-bipolar synaptic region. The guanylate cyclase is the previously characterized ROS-GC1 from the rod outer segments and its modulator is S100beta. S100beta senses increments in free Ca(2+) and stimulates the cyclase. Specificity of photoreceptor guanylate cyclase activation by S100beta is validated by the identification of two S100beta-regulatory sites. A combination of peptide competition, surface plasmon resonance binding and deletion mutation studies has been used to show that these sites are specific for S100beta and not for another regulator of ROS-GC1, guanylate cyclase-activating protein 1. One site comprises amino acids (aa) Gly962-Asn981, the other, aa Ile1030-Gln1041. The former represents the binding site. The latter binds S100beta only marginally, yet it is critical for control of maximal cyclase activity. The findings provide evidence for a new cyclic GMP transduction system in synaptic layers and thereby extend existing concepts of how a membrane-bound guanylate cyclase is regulated by small Ca(2+)-sensor proteins.