- Email: [email protected]
G-proteins and egg activation
Regulatory Mechanisms in Developmental Processes (G. Eguchi, 0 1988 Elsevier Scientific Publishers Ireland, Ltd.
T.S. Okada
and L. Sax&
eds.), 15-18
15
G-proteins and egg activation Laurinda
A. Jaffe ‘, Paul R. Turner 2, Douglas Kline ‘, Raymond and Fraser Shilling 4
T. Kado 3
’ Department of Physiology, University of Connecticut Health Center, Farmington, CT 06032, U.S.A.; ’ Department of Zoology, University of California, Berkeley, CA 94720, U.S.A.; ’ Luboratoire de Neurobiologie Cellulaire, C. N.R.S., Gif-sur- Yvette 91198, France; 4 Department of Biology, University of Southern California, University Park, Los Angeles, CA 90089, U.S.A.
G-proteins are present in eggs, and experiments in which GTPq-S, GDP-B-S, cholera toxin and pertussis toxin have been injected into eggs have indicated the involvement of G-proteins in egg activation at fertilization and in oocyte maturation. Eggs into which serotonin or muscarinic acetylcholine receptors have been introduced by mRNA injection produce fertilization-like responses when exposed to serotonin or acetylcholine; since these neurotransmitter receptors act by way of G-proteins, this observation further supports the conclusion that a G-protein is involved in the fertilization process. G-protein; Egg activation
Introduction
Guanine nucleotide binding proteins, or G-proteins, are a class of membrane proteins that act as intermediates between membrane receptors and effector proteins in signal transduction pathways (Stryer and Bourne, 1986; Gilman, 1987). G-proteins are known to mediate cellular responses to hormones, neurotransmitters and light; particular receptors are coupled to specific G-proteins, which are in turn coupled to particular effector enzymes (Fig. 1). G-proteins also appear to have a role in mediating the egg’s response to fertilization (Turner et al., 1988). The participation of a G-protein in a cellular process can be identified by a number of means, which include: (1) guanosine-5’-0-(3-thiotriphos-
Correspondence address: L.A. Jaffe, Dept. of Physiology, University of Connecticut Health Center, Farmington, CT 06032, U.S.A.
phate) (GTP-y-S), a hydrolysis-resistant analog of GTP, produces an irreversible activation of Gproteins. (2) Guanosine-5’-O-(2-thiodiphosphate) (GDP-P-S), a metabolically stable analog of GDP, inactivates G-proteins. (3) Cholera toxin (CTX) and pertussis toxin (PTX) catalyze the ADP-ribosylation of the a-subunits of certain G-proteins. If radioactive NAD is present with the toxins, labelling of the G-proteins results, allowing their detection. These toxins also affect the function of particular G-proteins; CTX causes irreversible activation, while PTX causes inactivation. Evidence will be presented in support of the hypothesis that sperm-egg interaction leads to the activation of a G-protein in the egg membrane, which in turn leads to the production of inositol trisphosphate (InsP,) and release of calcium. Calcium then initiates multiple responses of the egg to fertilization. Evidence will also be presented that a G-protein may be important in mediating the reinitiation of meiotic maturation of the starfish oocyte in response to 1-methyladenine.
16
G-proteins and egg activation at fertilization To look for G-proteins in sea urchin eggs, the labelling of egg proteins in the presence of CTX and PTX was examined. Oinuma et al. (1986) found a 39-kDa substrate for PTX, which copurified with the ability to bind GTP-y-S, as would be expected for a G-protein. Turner et al. (1987) reported that CTX catalyzed the ADP-ribosylation of a 47-kDa substrate, and PTX labelled a 40-kDa substrate. To test the hypothesis that a G-protein was a component of the pathway coupling sperm-egg interaction to exocytosis of cortical vesicles, sea urchin eggs were microinjected with GTP-y-S. In some, but not all trials, this caused exocytosis (Turner et al., 1986; L.A.J. and P.R.T., unpublished results). Measurements with the calcium indicator fura 2 showed that GTP-y-S injection caused a rise in intracellular free calcium (Swann
RECEPTOR G-PROTEIN Padrenergic serotonin muscarinic a-adrenergic
1 G, 3 1 G,
rhodopsin
6
muscarinic a-adrenergic serotonin 1
G,
EFFECTOR
adenylate cyclase
cGMPphosphodiesterase phospholipase A,
c
PIP, phosphodiesterase
Fig. 1. Receptor-G-protein-effector enzyme interaction in cells. The diagram illustrates the plasma membrane, with a receptor (R), G-protein (G) and effector enzyme (E). Arrows indicate the inhibitory (e) and stimulatory (e) actions of various agents: pertussis toxin (PTX), GDP-P-S, GTPq-S, and cholera toxin (CTX). Examples of pathways involving these components are listed. (Based on papers by Stryer and Boume, 1986; Dohlman et al., 1987; Gilman, 1987; Kobilka et al., 1987).
et al., 1988). Microinjection, but not external application of CTX or CTX subunit A, also resulted in exocytosis (Turner et al., 1987). The effects of both GTP-y-S and CTX injection were blocked by preinjecting the eggs with EGTA, suggesting that GTP-y-S and CTX stimulate exocytosis by causing an increase in calcium. This hypothesis was supported by the observation that microinjection of CAMP, or a hydrolysis-resistant analog of CAMP, did not cause exocytosis (Turner et al., 1987). If the activation of a G-protein is required for exocytosis, then inactivation of the G-protein should prevent the stimulation of exocytosis by sperm. Indeed, in eggs pre-injected with GDP-P-S, sperm did not stimulate exocytosis (Turner et al., 1986), although they did enter the eggs (Swann et al., 1988). If GDP-P-S injected eggs were subsequently injected with InsP,, exocytosis resulted, indicating that the step involving the CTX-sensitive G-protein preceded the step involving the InsP, (Turner et al., 1986). GTP-y-S injection also stimulated cortical vesicle exocytosis and an activation potential in frog eggs (Kline and Jaffe, 1987). In hamster eggs, GTP-y-S injection initiated an electrical response like that seen at fertilization, whereas GDP-P-S injection blocked this response to sperm (Miyazaki, 1988). To further investigate the possible function of G-proteins in fertilization of frog eggs, serotonin and Ml acetylcholine receptors, which are known to act by way of G-proteins (Dascal et al., 1986; Nomura et al., 1987), were introduced into the Xenopus egg (Kline et al., 1988). The serotonin and acetylcholine receptors were introduced by injection of mRNA into oocytes; the oocytes were then induced to mature by exposure to progesterone. In response to serotonin or acetylcholine, such eggs produced an activation potential and underwent cortical vesicle exocytosis. These responses to serotonin and acetylcholine were not observed in control non-injected eggs. These results suggest that the exogenously introduced serotonin and acetylcholine receptors interact with an endogenous G-protein in the frog egg membrane that is normally activated by sperm, and support the hypothesis that receptor-mediated
17
h
/
Coz+ RELEASE +
EXOCYlOslS
ornER MVELOPMENTAL EVENTS
Fig. 2. Possible involvement of a receptor-G-protein-effector enzyme pathway in the egg membrane at fertilization. Interaction of the sperm with a hypothetical ‘sperm receptor’ is proposed to lead to activation of a G-protein. The target of the G-protein may be PIP, phosphodiesterase, which hydrolyzes phosphatidylinositol bisphosphate (PIP,) to produce diacylglycerol (DAG) and inositol stores, leading to ion channel opening, exocytosis and other trisphosphate (InsP,). InsP, releases Ca 2+ from intracellular developmental events.
activation of a G-protein initiates the response of the egg to fertilization. Based on the experiments described above, we have proposed a model for the role of G-proteins in the activation of eggs at fertilization (Fig. 2). For further discussion of this model, see Turner and Jaffe (1988).
G-proteins and oocyte maturation G-proteins may also be important in mediating the reinitiation of meiosis in starfish oocytes, in response to 1-methyladenine (l-MA) (Shilling and Jaffe, 1987). l-MA is believed to act on a receptor on the external surface of the oocyte (Kanatani and Hiramoto, 1970; Yoshikuni et al., 1988) leading by way of unknown intermediates to the production of maturation promoting factor in the cytoplasm, which in turn causes germinal vesicle breakdown (GVBD) and reinitiation of meiosis (Kishimoto and Kanatani, 1976; Kishimoto and Kondo, 1986). To investigate the possible role of a G-protein in transducing the hormonal signal across the oocyte membrane, starfish oocytes were injected with GDP-P-S or PTX. Both inhibited GVBD in response to l-MA (Shilling and Jaffe, 1987; similar results have been obtained by K.
Chiba and M. Hoshi, personal communication). These observations indicated the involvement of a G-protein in the coupling of l-MA binding to the reinitiation of meiosis.
Receptor and effector proteins? In summary, these studies of G-proteins in gametes have identified their function in oocyte maturation, sperm activation (Endo et al., 1987) and egg activation. By comparison with other cellular systems known to involve a G-protein, these findings give some indications about the receptors and effector proteins that may be involved in these processes. All known receptors that interact with G-proteins have a common protein structure (Dohlman et al., 1987; Kobilka et al., 1987). This suggests that there may be related receptors for sperm and for 1-methyladenine. G-proteins activate a diverse group of effector proteins, some of which may also be involved in the activation of oocytes, eggs and sperm.
Acknowledgement This work was supported
by NIH.
18
References Dascal, N., C. Ifune, R. Hopkins, T.P. Snutch, H. Lubbert, N. Davidson, M.I. Simon and H.A. Lester: Involvement of a GTP-binding protein in mediation of serotonin and acetylcholine responses in Xenopus oocytes injected with rat brain messenger RNA. Mol. Brain Res. 1, 201-209 (1986). Dohlman, H.G., M.G. Caron and R.J. Lefkowitz: A family of receptors coupled to guanine nucleotide regulatory proteins, Biochemistry 26, 2657-2668 (1987). Endo, Y., M.A. Lee and G.S. Kopf: Evidence for the role of a guanine nucleotide-binding regulatory protein in the zona pellucida-induced mouse sperm acrosome reaction. Dev. Biol. 119, 210-216 (1987). Gilman, A.G.: G proteins: Transducers of receptor-generated signals. Annu. Rev. Biochem. 56, 615-649 (1987). Kanatani, H. and Y. Hiramoto: Site of action of l-methyladenine in inducing oocyte maturation in starfish. Exp. Cell Res. 61, 280-284 (1970). Kishimoto, T. and H. Kanatani: Cytoplasmic factor responsible for germinal vesicle breakdown and meiotic maturation in starfish oocyte. Nature 260, 321-322 (1976). Kishimoto, T. and H. Kondo: Extraction and preliminary characterization of maturation promoting factor from starfish oocytes. Exp. Cell Res. 163, 44-452 (1986). Kline, D. and L.A. Jaffe: The fertilization potential of the Xenopus egg is blocked by injection of a calcium buffer and is mimicked by injection of a GTP analog. Biophys. J. 51, 398a (1987). Kline, D., L. Simoncini, G. Mandel, R. Maue, R.T. Kado and L.A. Jaffe: Fertilization events induced by neurotransmitters after injection of mRNA into Xenopus eggs. Science, in press (1988). Kobilka, B.K., H. Matsui, T.S. Kobilka, T.L. Yang-Feng, U. Francke, M.G. Caron, R.J. Lefkowitz and J.W. Regan: Cloning, sequencing and expression of the gene coding for the human platelet n,-adrenergic receptor. Science 238, 650-656 (1987).
Miyazaki, S.: Inositol 1,4,5-trisphosphate-induced calcium release and GTP binding protein-mediated periodic calcium rises in golden hamster eggs. J. Cell Biol. 106, 345-353 (1988). Nomura, Y., S. Kaneko, K. Kato, S. Yamagishi and H. Sugiyama: Inositol phosphate formation and chloride current responses induced by acetylcholine and serotonin through GTP-binding proteins in Xenopus oocyte after injection of rat brain messenger RNA. Mol. Brain Res. 2, 113-123 (1987). Oinuma, M., T. Katada, H. Yokosawa and M. Ui: Guanine nucleotide-binding protein in sea urchin eggs serving as the specific substrate of islet-activating protein, pertussis toxin. FEBS Lett. 207, 28-34 (1986). Shilling, F. and L.A. Jaffe: Evidence that a G-protein mediates 1-methyladenine induced maturation of starfish oocytes. Biol. Bull. 173, 427 (1987). Stryer, L. and H.R. Bourne: G proteins: A family of signal transducers. Annu. Rev. Cell Biol. 2, 391-419 (1986). Swann, K., B. Ciapa and M. Whitaker: Cellular messengers and sea urchin egg activation. In: Molecular Biology of Invertebrate Development, ed. D. O’Connor (Alan R. Liss, New York) in press (1988). Turner, P.R. and L.A. Jaffe: G-proteins and the regulation of oocyte maturation and fertilization. In: The Cell Biology of Fertilization, eds. H. Schatten and G. Schatten (Academic Press, Orlando) in press (1988). Turner, P.R., L.A. Jaffe and A. Fein: Regulation of cortical vesicle exocytosis in sea urchin eggs by inositol 1,4,5-trisphosphate and GTP-binding protein. J. Cell Biol. 102, 70-76 (1986). Turner, P.R., L.A. Jaffe and P. Primakoff: A cholera toxin-sensitive G-protein stimulates exocytosis in sea urchin eggs. Dev. Biol. 120, 577-583 (1987). Yoshikuni, M., K. Ishikawa, M. Isobe, T. Goto and Y. Nagahama: Characterization of I-methyladenine binding in starfish oocyte cortices. Proc. Natl. Acad. Sci. USA 85, 187441877 (1988).
T.S. Okada
and L. Sax&
eds.), 15-18
15
G-proteins and egg activation Laurinda
A. Jaffe ‘, Paul R. Turner 2, Douglas Kline ‘, Raymond and Fraser Shilling 4
T. Kado 3
’ Department of Physiology, University of Connecticut Health Center, Farmington, CT 06032, U.S.A.; ’ Department of Zoology, University of California, Berkeley, CA 94720, U.S.A.; ’ Luboratoire de Neurobiologie Cellulaire, C. N.R.S., Gif-sur- Yvette 91198, France; 4 Department of Biology, University of Southern California, University Park, Los Angeles, CA 90089, U.S.A.
G-proteins are present in eggs, and experiments in which GTPq-S, GDP-B-S, cholera toxin and pertussis toxin have been injected into eggs have indicated the involvement of G-proteins in egg activation at fertilization and in oocyte maturation. Eggs into which serotonin or muscarinic acetylcholine receptors have been introduced by mRNA injection produce fertilization-like responses when exposed to serotonin or acetylcholine; since these neurotransmitter receptors act by way of G-proteins, this observation further supports the conclusion that a G-protein is involved in the fertilization process. G-protein; Egg activation
Introduction
Guanine nucleotide binding proteins, or G-proteins, are a class of membrane proteins that act as intermediates between membrane receptors and effector proteins in signal transduction pathways (Stryer and Bourne, 1986; Gilman, 1987). G-proteins are known to mediate cellular responses to hormones, neurotransmitters and light; particular receptors are coupled to specific G-proteins, which are in turn coupled to particular effector enzymes (Fig. 1). G-proteins also appear to have a role in mediating the egg’s response to fertilization (Turner et al., 1988). The participation of a G-protein in a cellular process can be identified by a number of means, which include: (1) guanosine-5’-0-(3-thiotriphos-
Correspondence address: L.A. Jaffe, Dept. of Physiology, University of Connecticut Health Center, Farmington, CT 06032, U.S.A.
phate) (GTP-y-S), a hydrolysis-resistant analog of GTP, produces an irreversible activation of Gproteins. (2) Guanosine-5’-O-(2-thiodiphosphate) (GDP-P-S), a metabolically stable analog of GDP, inactivates G-proteins. (3) Cholera toxin (CTX) and pertussis toxin (PTX) catalyze the ADP-ribosylation of the a-subunits of certain G-proteins. If radioactive NAD is present with the toxins, labelling of the G-proteins results, allowing their detection. These toxins also affect the function of particular G-proteins; CTX causes irreversible activation, while PTX causes inactivation. Evidence will be presented in support of the hypothesis that sperm-egg interaction leads to the activation of a G-protein in the egg membrane, which in turn leads to the production of inositol trisphosphate (InsP,) and release of calcium. Calcium then initiates multiple responses of the egg to fertilization. Evidence will also be presented that a G-protein may be important in mediating the reinitiation of meiotic maturation of the starfish oocyte in response to 1-methyladenine.
16
G-proteins and egg activation at fertilization To look for G-proteins in sea urchin eggs, the labelling of egg proteins in the presence of CTX and PTX was examined. Oinuma et al. (1986) found a 39-kDa substrate for PTX, which copurified with the ability to bind GTP-y-S, as would be expected for a G-protein. Turner et al. (1987) reported that CTX catalyzed the ADP-ribosylation of a 47-kDa substrate, and PTX labelled a 40-kDa substrate. To test the hypothesis that a G-protein was a component of the pathway coupling sperm-egg interaction to exocytosis of cortical vesicles, sea urchin eggs were microinjected with GTP-y-S. In some, but not all trials, this caused exocytosis (Turner et al., 1986; L.A.J. and P.R.T., unpublished results). Measurements with the calcium indicator fura 2 showed that GTP-y-S injection caused a rise in intracellular free calcium (Swann
RECEPTOR G-PROTEIN Padrenergic serotonin muscarinic a-adrenergic
1 G, 3 1 G,
rhodopsin
6
muscarinic a-adrenergic serotonin 1
G,
EFFECTOR
adenylate cyclase
cGMPphosphodiesterase phospholipase A,
c
PIP, phosphodiesterase
Fig. 1. Receptor-G-protein-effector enzyme interaction in cells. The diagram illustrates the plasma membrane, with a receptor (R), G-protein (G) and effector enzyme (E). Arrows indicate the inhibitory (e) and stimulatory (e) actions of various agents: pertussis toxin (PTX), GDP-P-S, GTPq-S, and cholera toxin (CTX). Examples of pathways involving these components are listed. (Based on papers by Stryer and Boume, 1986; Dohlman et al., 1987; Gilman, 1987; Kobilka et al., 1987).
et al., 1988). Microinjection, but not external application of CTX or CTX subunit A, also resulted in exocytosis (Turner et al., 1987). The effects of both GTP-y-S and CTX injection were blocked by preinjecting the eggs with EGTA, suggesting that GTP-y-S and CTX stimulate exocytosis by causing an increase in calcium. This hypothesis was supported by the observation that microinjection of CAMP, or a hydrolysis-resistant analog of CAMP, did not cause exocytosis (Turner et al., 1987). If the activation of a G-protein is required for exocytosis, then inactivation of the G-protein should prevent the stimulation of exocytosis by sperm. Indeed, in eggs pre-injected with GDP-P-S, sperm did not stimulate exocytosis (Turner et al., 1986), although they did enter the eggs (Swann et al., 1988). If GDP-P-S injected eggs were subsequently injected with InsP,, exocytosis resulted, indicating that the step involving the CTX-sensitive G-protein preceded the step involving the InsP, (Turner et al., 1986). GTP-y-S injection also stimulated cortical vesicle exocytosis and an activation potential in frog eggs (Kline and Jaffe, 1987). In hamster eggs, GTP-y-S injection initiated an electrical response like that seen at fertilization, whereas GDP-P-S injection blocked this response to sperm (Miyazaki, 1988). To further investigate the possible function of G-proteins in fertilization of frog eggs, serotonin and Ml acetylcholine receptors, which are known to act by way of G-proteins (Dascal et al., 1986; Nomura et al., 1987), were introduced into the Xenopus egg (Kline et al., 1988). The serotonin and acetylcholine receptors were introduced by injection of mRNA into oocytes; the oocytes were then induced to mature by exposure to progesterone. In response to serotonin or acetylcholine, such eggs produced an activation potential and underwent cortical vesicle exocytosis. These responses to serotonin and acetylcholine were not observed in control non-injected eggs. These results suggest that the exogenously introduced serotonin and acetylcholine receptors interact with an endogenous G-protein in the frog egg membrane that is normally activated by sperm, and support the hypothesis that receptor-mediated
17
h
/
Coz+ RELEASE +
EXOCYlOslS
ornER MVELOPMENTAL EVENTS
Fig. 2. Possible involvement of a receptor-G-protein-effector enzyme pathway in the egg membrane at fertilization. Interaction of the sperm with a hypothetical ‘sperm receptor’ is proposed to lead to activation of a G-protein. The target of the G-protein may be PIP, phosphodiesterase, which hydrolyzes phosphatidylinositol bisphosphate (PIP,) to produce diacylglycerol (DAG) and inositol stores, leading to ion channel opening, exocytosis and other trisphosphate (InsP,). InsP, releases Ca 2+ from intracellular developmental events.
activation of a G-protein initiates the response of the egg to fertilization. Based on the experiments described above, we have proposed a model for the role of G-proteins in the activation of eggs at fertilization (Fig. 2). For further discussion of this model, see Turner and Jaffe (1988).
G-proteins and oocyte maturation G-proteins may also be important in mediating the reinitiation of meiosis in starfish oocytes, in response to 1-methyladenine (l-MA) (Shilling and Jaffe, 1987). l-MA is believed to act on a receptor on the external surface of the oocyte (Kanatani and Hiramoto, 1970; Yoshikuni et al., 1988) leading by way of unknown intermediates to the production of maturation promoting factor in the cytoplasm, which in turn causes germinal vesicle breakdown (GVBD) and reinitiation of meiosis (Kishimoto and Kanatani, 1976; Kishimoto and Kondo, 1986). To investigate the possible role of a G-protein in transducing the hormonal signal across the oocyte membrane, starfish oocytes were injected with GDP-P-S or PTX. Both inhibited GVBD in response to l-MA (Shilling and Jaffe, 1987; similar results have been obtained by K.
Chiba and M. Hoshi, personal communication). These observations indicated the involvement of a G-protein in the coupling of l-MA binding to the reinitiation of meiosis.
Receptor and effector proteins? In summary, these studies of G-proteins in gametes have identified their function in oocyte maturation, sperm activation (Endo et al., 1987) and egg activation. By comparison with other cellular systems known to involve a G-protein, these findings give some indications about the receptors and effector proteins that may be involved in these processes. All known receptors that interact with G-proteins have a common protein structure (Dohlman et al., 1987; Kobilka et al., 1987). This suggests that there may be related receptors for sperm and for 1-methyladenine. G-proteins activate a diverse group of effector proteins, some of which may also be involved in the activation of oocytes, eggs and sperm.
Acknowledgement This work was supported
by NIH.
18
References Dascal, N., C. Ifune, R. Hopkins, T.P. Snutch, H. Lubbert, N. Davidson, M.I. Simon and H.A. Lester: Involvement of a GTP-binding protein in mediation of serotonin and acetylcholine responses in Xenopus oocytes injected with rat brain messenger RNA. Mol. Brain Res. 1, 201-209 (1986). Dohlman, H.G., M.G. Caron and R.J. Lefkowitz: A family of receptors coupled to guanine nucleotide regulatory proteins, Biochemistry 26, 2657-2668 (1987). Endo, Y., M.A. Lee and G.S. Kopf: Evidence for the role of a guanine nucleotide-binding regulatory protein in the zona pellucida-induced mouse sperm acrosome reaction. Dev. Biol. 119, 210-216 (1987). Gilman, A.G.: G proteins: Transducers of receptor-generated signals. Annu. Rev. Biochem. 56, 615-649 (1987). Kanatani, H. and Y. Hiramoto: Site of action of l-methyladenine in inducing oocyte maturation in starfish. Exp. Cell Res. 61, 280-284 (1970). Kishimoto, T. and H. Kanatani: Cytoplasmic factor responsible for germinal vesicle breakdown and meiotic maturation in starfish oocyte. Nature 260, 321-322 (1976). Kishimoto, T. and H. Kondo: Extraction and preliminary characterization of maturation promoting factor from starfish oocytes. Exp. Cell Res. 163, 44-452 (1986). Kline, D. and L.A. Jaffe: The fertilization potential of the Xenopus egg is blocked by injection of a calcium buffer and is mimicked by injection of a GTP analog. Biophys. J. 51, 398a (1987). Kline, D., L. Simoncini, G. Mandel, R. Maue, R.T. Kado and L.A. Jaffe: Fertilization events induced by neurotransmitters after injection of mRNA into Xenopus eggs. Science, in press (1988). Kobilka, B.K., H. Matsui, T.S. Kobilka, T.L. Yang-Feng, U. Francke, M.G. Caron, R.J. Lefkowitz and J.W. Regan: Cloning, sequencing and expression of the gene coding for the human platelet n,-adrenergic receptor. Science 238, 650-656 (1987).
Miyazaki, S.: Inositol 1,4,5-trisphosphate-induced calcium release and GTP binding protein-mediated periodic calcium rises in golden hamster eggs. J. Cell Biol. 106, 345-353 (1988). Nomura, Y., S. Kaneko, K. Kato, S. Yamagishi and H. Sugiyama: Inositol phosphate formation and chloride current responses induced by acetylcholine and serotonin through GTP-binding proteins in Xenopus oocyte after injection of rat brain messenger RNA. Mol. Brain Res. 2, 113-123 (1987). Oinuma, M., T. Katada, H. Yokosawa and M. Ui: Guanine nucleotide-binding protein in sea urchin eggs serving as the specific substrate of islet-activating protein, pertussis toxin. FEBS Lett. 207, 28-34 (1986). Shilling, F. and L.A. Jaffe: Evidence that a G-protein mediates 1-methyladenine induced maturation of starfish oocytes. Biol. Bull. 173, 427 (1987). Stryer, L. and H.R. Bourne: G proteins: A family of signal transducers. Annu. Rev. Cell Biol. 2, 391-419 (1986). Swann, K., B. Ciapa and M. Whitaker: Cellular messengers and sea urchin egg activation. In: Molecular Biology of Invertebrate Development, ed. D. O’Connor (Alan R. Liss, New York) in press (1988). Turner, P.R. and L.A. Jaffe: G-proteins and the regulation of oocyte maturation and fertilization. In: The Cell Biology of Fertilization, eds. H. Schatten and G. Schatten (Academic Press, Orlando) in press (1988). Turner, P.R., L.A. Jaffe and A. Fein: Regulation of cortical vesicle exocytosis in sea urchin eggs by inositol 1,4,5-trisphosphate and GTP-binding protein. J. Cell Biol. 102, 70-76 (1986). Turner, P.R., L.A. Jaffe and P. Primakoff: A cholera toxin-sensitive G-protein stimulates exocytosis in sea urchin eggs. Dev. Biol. 120, 577-583 (1987). Yoshikuni, M., K. Ishikawa, M. Isobe, T. Goto and Y. Nagahama: Characterization of I-methyladenine binding in starfish oocyte cortices. Proc. Natl. Acad. Sci. USA 85, 187441877 (1988).