Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations

2004 The identification, cloning, and characterization of a Phospholipase C-beta isoform from the sea urchin egg Andre Peter Joseph Kulisz Iowa State University

Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Cell Biology Commons

Recommended Citation Kulisz, Andre Peter Joseph, "The identification, cloning, and characterization of a Phospholipase C-beta isoform from the sea urchin egg " (2004). Retrospective Theses and Dissertations. 1105. https://lib.dr.iastate.edu/rtd/1105

This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The Identification, cloning, and characterization of a Phospholipase C-beta isoform from the sea urchin egg

by

Andre Peter Joseph Knlisz

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Zoology

Program of Study Committee: Sheldon S. Shen, Major Professor Charles D. Drewes Michael McCloskey F. Anderson Norris Jo Anne Powell-CofBnan

Iowa State University

Ames, Iowa

2004

Copyright © Andre Peter Joseph Kulisz, 2004. All rights reserved. UMI Number: 3145665

INFORMATION TO USERS

The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion.

UMI

UMI Microform 3145665 Copyright 2004 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.

ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 11

Graduate College Iowa State University

This is to certify that the doctoral dissertation of

Andre Peter Joseph Kulisz has met the dissertation requirements of Iowa State University

Signature was redacted for privacy.

Major Professor

Signature was redacted for privacy. For the Major Pce iii

TABLE OF CONTENTS

LIST OF FIGURES iv

ABSTRACT v

CHAPTER 1. INTRODUCTION 1

CHAPTER 2. BACKGROUND 8

CHAPTERS. MATERIALS AND METHODS 20

CHAPTER 4. RESULTS 31

CHAPTER 5. DISCUSSION AND CONCLUSIONS 58

APPENDIX 63

REFERENCES 104 iv

LIST OF FIGURES

Figure 1. PGR products ofPLCp from sea urchin. 43

Figure 2. Amino acid alignment of partial sea urchin PLC(S with known 44

PLCp sequences.

Figure 3. Amino acid sequence of sea urchin PLCp. 45

Figure 4. Comparison of suPLCp to other known PLCp sequences. 46

Figure 5. Generation of a full-length suPLCp clone. 47

Figure 6. Anti-suPLCp Western blot analysis of protein lysates. 48

Figure 7. Anti-suPLC(3 Western blot analysis of vonegofwf 49

protein lysates.

Figure 8. Comparison of Zyfec&mwssuPLCp with 50

/wrpwrnAt; genome database.

Figure 9. Recombinant suPLCP expression and puiiûcation. 51

Figure 10. Gpy dose-response curve for suPLCp activation. 52

Figure 11. Calcium dependence of suPLCP activity. 53

Figure 12. Thrombin-mediated proteolysis of GST-suPLCp. 54

Figure 13. G|3y dose-response curve for activation of truncated suPLCp. 55

Figure 14. Calcium dependence of truncated suPLCP activity. 56

Figure 15. Potential G^y-interaction site in suPLCp. 57 V

ABSTRACT

This work was the first to provide direct evidence for the presence of a PLCP isofbrm, named suPLCp, within the sea urchin egg. The coding sequence was identified and cloned from the eggs of Z/yfecAmwa and when translated, determined to have the greatest degree of homology and identity with the mammalian PLC^4 isofbrms. This alone is a unique finding in that the presence of PLCp4 has only been found in the retina and specific neuronal tissues. The presence of the protein within the egg was verified using

antibodies that were generated specifically against suPLCp, indicating that the majority of

the enzyme is localized in the non-soluble fraction of the cell, presumably associated with the

plasma membrane. This distribution is found both in the unfertilized eggs as well as one

minute post-fertilization. In order to further characterize the enzymatic activity of suPLCp,

recombinant proteins were expressed and purified from

Initial experiments were carried out with the fusion protein bound to Glutathione-Sepharose

beads using the level of hydrolytic activity of the PLC as a measure of activity. Unlike

PLCp4, suPLCp is activated by G protein Py subunits, and this activity is dependent on the

levels of free calcium that are present. Removal of the GST tag via Thrombin-mediated

proteolysis not only freed the recombinant suPLCp from the Sepharose beads, but also

resulted in a truncated form of the protein that was missing the first 93 amino terminal amino

acid residues. When assayed for its enzymatic activity, this truncated form still possessed its

calcium-dependent hydrolytic activity that was regulated by G|3y. 1

CHAPTER 1. INTRODUCTION

Fertilization is the process whereby the egg and sperm fuse together; creating a new individual with a genetic potential that is derived from both parents. In addition, this process triggers several biochemical events that stimulate egg metabolism, activate biosynthetic pathways, and initiate cell division. All of these events are central for normal development.

An early universal event of animal fertilization is an increase in intracellular Ca^ activity

(Jaffe, 1991). Despite the importance of this event, the direct link between fertilization by sperm and this increase in Ca^ activity is still not clear (Strieker, 1999).

Ca^ is a ubiquitous intracellular signaling molecule controlling a wide array of cellular processes that include secretion, contraction, and cell proliferation (Berridge, 1993;

Clapham, 1995). In resting cells, the intracellular Ca^ concentration ([Ca^];) is maintained at approximately 50-200 nM and represents only a minute fraction of the total cellular Ca^ content, so the bulk of the cellular Ca^ is bound or resides in membrane-bound organelles

(Carfbli, 1987). During stimulation the [Ca^]; may increase up to several micromolar and, may have complex timing and localization depending on the cell type. The mechanisms that underlie these events are not entirely clear. In many cell types, a sustained increase in [Ca^], does not occur, but instead is presented in the cytoplasm in a pulsatile manner, such as the repetitive Ca^ spikes that drive the beating heart, the rapid subplasmalemmal [Ca^]j increases that occur during depolarization of excitable cells, and the regular [Ca^]; oscillations that are observed during hormonal stimulation of many non-excitable cells.

Since information is encoded in the spatiotemporal patterns of these intracellular signals 2

(Clapham, 1995), an understanding of how these complex Ca^ signals are generated is of great importance.

There are two different sources that can contribute to the increase in [Ca^], within a given cell. The first source is an influx of Ca^ from the extracellular environment through ion channels or exchangers present in the plasma membrane of the cell. The second source is through mechanisms that release intracellular (sarcoplasmic/endoplasmic) stores of Ca^, and thus elevate [Ca2+]j. Release of Ca2+ from organelles is generally dependent on one of two types of intracellular receptor release mechanisms, sensitive to either the vegetal alkaloid ryanodine or to inositol 1,4,5-trisphosphate (IP3) (Berridge, 1993). Recently, a potential third

Ca2+-release second messenger has been identified. Nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to mobilize intracellular Ca^ stores in invertebrate, lower vertebrate, plant, and mammalian cells (Chini and Toledo, 2002; Genazzani and

Billington, 2002).

The physiological agonist of the ryanodine receptor has not yet been identified, but it is thought to be cyclic ADP-ribose (cADPR) (Galione, 1993a, 1993b; Lee, 1993). It has been reported that cADPR is capable of mobilizing Ca^ in various cell types including pituitary cells (Koshiyama ef a/., 1991), dorsal root ganglion cells (Currie ef a/., 1992), pancreatic p cells (Takasawa ef a/., 1993), cardiac sarcoplasmic reticulum microsomes

(Mészàros ef oZ., 1993), and rat brain microsomes (White ef a/., 1993). Synthesis of cADPR

&om p-NAD^ may occur by either the cytosolic enzyme, ADP-ribosyl cyclase (Lee and

Aarhus, 1991), or by the membrane bound enzyme, NAD glycohydrolase (Kim ef a/., 1993). 3

Degradation of cADPR to ADPR by cADPR-ribose hydrolase is a part of NAD glycohydrolase activity (Kim ef a/., 1993) or ADP-ribosyl cyclase activity (Zocchi ef a/.,

1993). Furthermore, cADPR and its synthesizing enzyme have been detected in a wide variety of mammalian and invertebrate cell types (Rusinko and Lee, 1989; Walseth ef a/.,

1991; Lee and Aarhus, 1991). In the sea urchin egg, the enzyme is associated with the membrane fraction, but is still referred to as ADP-ribosyl cyclase (Lee and Aarhus, 1991).

ADP-ribosyl cyclase can be activated by a cGMP-dependant kinase (PKG), as demonstrated in PC12 cells (Clementi ef a/., 1996). The generation of the intracellular second messenger signaling molecule, 3',5'-cyclic guanosine monophosphate (cGMP) and pyrophosphate, is initiated by the production of nitric oxide (NO) via nitric oxide synthase

(NOS) (reviewed by Hanafy ef a/., 2001). There are three isofbrms of the NOS enzyme: neuronal NOS (nNOS), epithelial NOS (eNOS), and inducible NOS (iNOS). NOS forms homodimers which catalyze the formation of L-citrulline and NO from L-arginine using oxygen and NADPH as cosubstrates. Of the three enzymes, iNOS is the only one that does not require elevated Ca^ for activation. Soluble guanylyl cyclase (sGC) is a heterodimer composed of an a and a (3 subunit. Upon binding of NO, sGC catalyzes the conversion of guanosine 5'-triphosphate (GTP) to cGMP.

IP3 and diacylglycerol (DAG) are generated as a result of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) through the enzymatic activity of phospholipase

C (PLC). Currently, there are five known isozymes within the PLC family of enzymes:

PLCP, PLCy, PLCÔ, PLCe and PLC{". Numerous studies have focused on the regulation of 4 these isozymes. PLCs contains the conserved catalytic (X and Y) and regulatory (C2) domains present in all PLC isozymes, but it is unique in containing a Ras guanine nucleotide exchange factor (CDC25)-like domain and at least one (sometimes two) Ras binding (RA)

domains (Lopez ef oA, 2001; Song ef aA, 2001). The presence of these two unique domains

suggests that PLCs may be an effector of Ras. It has also been shown that PLCs can be

activated by Ga#, while the region of subunit interaction with the enzyme is unknown (Song

gf a/., 2001). A previously unrecognized pleckstrin homology (PH) domain followed by an

EF-hand domain was identified in the amino terminus of PLCs. Based on this information, it

was further demonstrated that heterotrimeric G protein py-subunits were capable of

stimulating PLCs catalytic activity (Wing ef a/., 2001). These Endings within the enzyme's

sequence revealed the presence of additional functional domains and added a new level of

complexity in the regulation of this novel enzyme.

The mechanism by which PLC8 is activated is still unclear, with some evidence

implicating the GTPase-activating protein RhoGAP (Homma and Emori, 1995) or the GTP-

binding protein Gh (Feng ef oA, 1996) in its activation. It has also been shown that PLC6 is

regulated by Ca2* and therefore may serve to potentiate intracellular Ca^ activity after the

initial increase is generated by one of the other PLC family members.

PLC was initially identified in the mouse EST database with expression derived

from a single testis gene (Saunders ef aA, 2002). The complete cDNA sequence was cloned

from a mouse spermatid cDNA library, and upon sequence analysis, appears to be a novel

PLC isofbrm that is smaller than the other four identified PLC family members. Multiple

amino acid sequence alignment with the other mammalian PLC isofbrms shows that it has 5 the highest degree of similarity with the PLC8 group, the main differences being that PLCf lacks an N-terminal PH domain and contains an extended X-Y linker sequence with a high proportion of charged residues. Western blot analysis revealed that this unique PLC is present specifically in whole sperm and in sperm extracts from mouse, hamster, pig, and humans (Swann gf a/., 2004). When sperm extracts were fractionated and assayed for the

ability to induce Ca^ release in sea urchin homogenates, the activity was correlated with

fractions containing PLCS" (Saunders ef a/., 2002). In addition to being highly effective in

causing Ca^ oscillations when injected into mouse eggs, PLCÇ is also an effective

parthenogenetic activating agent for mouse eggs (Swann et al., 2004).

The activity of PLCy is regulated by the association of its tandem src-homology 2

(SH2) domains with the specific phosphorylated tyrosine residues of a receptor tyrosine

kinase, which phosphorylates PLCy, thereby activating it (Rhee and Bae, 1997; Rebecchi and

Pentyala, 2001; Rhee, 2001). Recent studies have proposed an alternative model of PLCy

activation. Phosphatidylinositol 3,4,5-trisphosphate (PIP3), the product of the enzyme

phosphoinositide 3-kinase (PI 3-kinase) acting on PIP2, can potentially increase IP3

production and Ca^ release by recruiting and/or stabilizing PLCy to the lipid bilayer thereby

increasing PIP2 substrate availability. PIP3 binds with the PH domain of PLCy and targets the

enzyme to the membrane in response to growth factor stimulation (Falasca ef a/., 1998). An

interaction of PIP3 with the SH2 domain has also been demonstrated leading to the activation

of PLCy in the absence of tyrosine phosphorylation (Bae ef a/., 1998; Rameh ef 0/., 1998).

The Oq/n or Py subunits of heterotrimeric G proteins activate PLCP (Rhee and Bae,

1997; Rebecchi and Pentyala, 2001; Rhee, 2001). The regulation of this activity is based on 6 interactions of each of the G protein subunits with the different structural regions of the enzyme. At the N-terminus region there is a PH domain, followed by four EF-hand motifs just before the catalytic core domain (X and Y domains) of the enzyme. A C2 domain is next followed by an extended C-terminal sequence, which is unique to PLCp. The PH domain has a high membrane binding affinity and has been suggested to act as an anchor to the membrane surface allowing for lateral association with G proteins (Wang gf aA, 1999a). It has also been shown that the PH domain has a high affinity for the G|3y dimer and therefore may act as a docking site for activation of the catalytic domain (Wang ef a/., 1999a; Wang gf aA, 2000). The extended COOH-terminal domain is important for Goq interactions. This was demonstrated with removal of the region in PLCP-1 by calpain, a Ca^-dependent protease, which abolished activation by Goq (Park ef a/., 1993) as well as by constructing deletion mutants that were missing specific sequence residues in the C-terminal end of the protein (Wu gf aA, 1993). C2 domains are found in various proteins and in most cases bind

Ca^. These domains are also capable of membrane targeting. Interestingly, in PLC(3-1 and

PLCP-2 the C2 domain does not bind to the membrane; however, it selectively associates with activated Goq (Gctq -GTP) subunits (Wang gf aA, 1999b).

A&L4DP-jwg(#ofg(f rgfga&g

NAADP is a pyridine nucleotide derivative of NADP (nicotinamide adenine dinucleotide phosphate), that can induce Ca2* release in various cellular models ranging from marine invertebrates to mammals (Lee and Aarhus, 2000; Lim gf aA, 2001; Patel gf a/, 2001).

In the best characterized model for this molecule, the sea urchin egg, NAADP-mediated Ca2* 7 release is insensitive to inhibitors that act on the cADPR and IP3 pathways for Ca^ release

(Clapper ef oA, 1987; Chini ef oA, 1995; Lee and Aarhus, 1995). There are two other unique properties to the NAADP-mediated Ca^ release. First, the store utilized for Ca^ release is not the endoplasmic reticulum, as with cADPR and IP3 (Genazzani and Galione, 1996; Lee and Aarhus, 2000). Second, subthreshold concentrations of NAADP that do not induce Ca^ release block any further response to the messenger even at maximal concentrations (Aarhus

ef oA, 1996; Genazzani ef a/., 1996). Although it is established that an NAADP-mediated

Ca2+ release is present in various cell types, research is currently being done in order to

figure out the exact mechanism of this release and what its physiological significance

actually is. 8

CHAPTER 2. BACKGROUND

frxcreose fn /Co*"/; /krdKzaA)*

Of the two general sources for generating an increase in [Ça2*],, the influx of Ca2^ ions can be eliminated as a necessary signal for egg activation for the following reasons.

Fertilizing eggs in the absence of extracellular Ca^ does not prevent the rise in [Ca^]j

(Steinhardt and Epel, 1974; Schmidt ef a/., 1982; Fujino ef a/., 1985). The use of various pharmacological plasma membrane Ca^ channel blockers, which block Ca^ influx, also fail to prevent the fertilization-induced increase in [Ca^]; (Fujino ef a/., 1985; Fujiwara ef a/.,

1988; Shen and Buck, 1993; Strieker ef a/., 1994). Thus an intracellular release mechanism

is responsible for generating the increase in [Ca2+]j.

Based on data obtained from several species ranging from jellyfish to mammals, the fertilization-induced increase in [Ca^]j is due to the release of Ca2^ from the endoplasmic reticulum (ER) (reviewed by Shen, 1995; Jones, 1998; Strieker, 1999). The sea urchin egg contains three identified mechanisms for the release of intracellular Ca2\ which are regulated by IP3, cADPR, and NAADP. The pathway for the NAADP release mechanism is still unknown and, due to its unique inactivation properties (Genazzani ef a/., 1996), may be omitted from the list of possible Ca2^-release mechanisms that are activated during fertilization in the sea urchin egg. Proteins recognized by antibodies against receptors for the other two release pathways have been identified in sea urchin eggs. An antibody against the skeletal muscle type 1 ryanodine receptor recognizes a protein -380 kDa, which is

significantly smaller than the somatic receptor isofbrms (McPherson ef o/., 1992). An

antibody against the type IIP3 receptor recognizes a protein -370 kDa in size, which is larger 9 than the IP3 receptor isofbrms in rabbit brain and Aenopws eggs (Parys gf a/., 1994). Both receptor release mechanisms appear to utilize a common Ca2* store in the unfertilized egg

(Whalley gf aA, 1992; Buck gf 0/., 1994). In addition, molecular cloning and characterization of a ryanodine receptor from the unfertilized eggs of the sea urchin #gn»cgMfrof%s

^w/cAernmwa suggests a potential target for cADPR-induced Ca^ release (Shiwa gf aA,

2002).

refeaae af yêrfifkafww

Addition of cADPR (0.1 JJM) to sea urchin egg homogenates induced the release of

Ca2^ from internal stores (Dargie gf aA, 1990). It has been further demonstrated that microinjection of cADPR into intact sea urchin eggs is capable of inducing a Ca2* response similar to the fertilization-induced response (Dargie gf aA, 1990; Shen and Buck, 1993). In these studies, the threshold concentration of microinjected cADPR that is required to elicit the Ca2* response is 4 nM. The amount of cADPR produced during fertilization has been quantified in two species of Japanese sea urchin, #g#»cg»frof%? jWc&grnmws and vdMf&ocK&zri? cra&rwpiMa (Kuroda gf aA, 2001). In both species, the cADPR contents rose to

-5-15 amoles (10"^) per egg, peaked and subsided essentially in parallel with cGMP, at least during the first minute after fertilization.

At saturating concentration (0.6 pM), cADPR triggered a Ca2^ response with signiGcantly faster rise and fall times compared to the fertilization response (Shen and Buck,

1993). This finding does not eliminate the possibility of ryanodine receptor-dependent Ca2^ release during fertilization. Recently fertilized eggs are refractory to ryanodine (Buck gf aA,

1992) or cADPR (Shen and Buck, 1993), which is similar to the finding that Ca2^ release is 10 refractory for 20 minutes after fertilization (Zucker gf of., 1978). In an egg homogenate preparation, a sub-threshold level of cADPR activated ryanodine receptor-mediated Ca^ release with increasing Ca2* levels (Lee, 1993). In the intact sea urchin egg, full release of

Ca^ by cADPR requires a positive feedback by Ca2^ (Guo and Becker, 1997). Based on

these results, perhaps the cADPR-ryanodine receptor pathway for release of Ca2^ acts

synergistically with another Ca^ release pathway, and thus contributes in generating the bulk

rise in intracellular Ca2^ during fertilization.

The level of cADPR in sea urchin eggs may be regulated by a cGMP-dependent

pathway (Galione et al., 1993b). It has been proposed that cGMP, through cGMP-dependent

protein kinase (PKG), regulates the activity of ADP ribosyl cyclase, which in turn regulates

both cADPR synthesis from (3-NAD+ and cADPR hydrolysis to ADPR. The amount of

cGMP was reported to double in eggs during the first 10-20 seconds of fertilization (Ciapa

and Epel, 1996; Kuroda et al., 2001). Microinjection of 10-20 pM cGMP into sea urchin

eggs triggered inositol phospholipid-independent Ca2+ release (Whalley et al., 1992),

however, these values appear to be in great excess of the reported concentrations of -10

amoles (10"^ moles) cGMP present in the egg after insemination (Kuroda gf a/., 2001).

Using PKG inhibitors, H8 and Rp-8-pCPT-cGMPs, and ADP-ribosyl cyclase

inhibitors, nicotinamide and 3-amino-pyridine NAD\ it was confirmed that the Ca^

response to microinjection of cGMP was due to stimulation of cADPR production in intact

eggs (Lee gf a/., 1996). None of the mentioned PKG or ADP-ribosyl cyclase inhibitors

blocked Ca^ release in sea urchin eggs during fertilization (Lee gf a/., 1996). Kuroda and

colleagues (2001), using an inhibitor of soluble guanylate cyclase, 6-anilino-5, 8

quinolinedione (LY83583), confirmed formation of a fertilization envelope (a morphological 11 indicator of the rise in [Ca^], (Whitaker and Irvine,1984)) in inhibitor-treated eggs. The inhibitor could reduce the increase in [Ca^]j only at high concentrations (>100 pM), but had no effect on the latent periods for the [Ca^]; rise. All of the aforementioned inhibitors block cleavage in the fertilized egg.

Application of exogenous nitric oxide (NO) mobilizes Ca^ from intracellular stores in sea urchin eggs possibly by elevating cADPR levels in egg homogenates (Willmott gf a/.,

1996). Through the use of various pharmacological inhibitors of signaling intermediates, the

authors demonstrated that the NO acted through a signaling pathway involving cGMP and

led to the activation of a cADPR-sensitive Ca^ release mechanism. These results, although

using an exogenous stimulus, link cGMP and cADPR to a common Ca^-release signaling

pathway that is independent of IPg-mediated release. A new hypothesis had been proposed

implicating a role for NO in sea urchin egg activation at fertilization (Kuo gf aA, 2000). The

authors suggest that the endogenous generation of NO via a nitric oxide synthase (NOS in the

sperm) may lead to the direct release of Ca2* by S-nitrosylation of ryanodine-receptor stores

which in turn could lead to an additional Ca2^ release via a calcium-sensitive PLC generating

IPs. The authors further suggest that nitrosylation, acting as a method of post-translational

modification (analogous to protein phosphorylation), may modify signaling intermediates

such as arc which can then induce PLC activity. Based on this hypothesis that NO is a

primary egg activator during fertilization, two independent groups investigated the link

between NO and Ca2* in mouse and ascidian (Ascw&gffa ojperya) eggs (Hyslop gf a/., 2001)

and sea urchin (R%zmmgcAm%? /m/wzris and Z/yfgc/»M%? /xcfwa) eggs (Leckie gf oA, 2003) by

monitoring NO and Ca2* simultaneously with fluorescent indicators for each. Hyslop gf a/.

(2001) demonstrated that NO has no role in the fertilization of the two chordate eggs that 12 they investigated. Leckie ef a/. (2003) established that NO levels rise after, not before, the

Ca^ wave is initiated and that this rise is Ca^-dependent in sea urchin eggs. However, they suggest NO may act after the initiation of the Ca^ wave to regulate the duration of the fertilization Ca2+ transient.

TPj-maf&zfed refease of /èrdMW&w;

A rapid turnover of phosphatidylinositides has been observed in sea urchin eggs

(Turner et al., 1984; Ciapa et al., 1996) as well as an increase in the relative amounts of IP3 and DAG during fertilization (Ciapa and Whitaker, 1986). In sea urchin eggs, an increase in the mass of IP3 has been detected during the first 2 minutes of fertilization (Lee and Shen,

1998; Kuroda ef a/., 2001). It was observed that an initial increase in IP3 mass, detected -30 seconds after fertilization, preceded the rise in [Ca2*],. This initial increase in IP3 was then followed by a second increase, representing the bulk of the increase. The two-phased rise in

IP3 mass during fertilization was blocked in eggs pretreated with the PLC(5 inhibitor, U73122

(Lee and Shen, 1998). By monitoring fertilization envelope (FE) formation, it was determined that the initially small, yet significant rise in IP3 mass preceded FE formation, while the bulk of IP3 production occurred after the increase in [Ca^];.

Complete inhibition of the increase in [Ca2*]; during fertilization has been demonstrated in eggs pretreated with the IP3 receptor antagonists, heparin or pentosan polysulfate (Mohri ef aA, 1995). Using the PLCP inhibitor U73122 in order to block the synthesis of IP3, Lee and Shen (1998) were able to inhibit the sperm-induced increase in

[Ca^]i and cytoplasmic alkalinization in a dose-dependent manner. An increase in intracellular pH during fertilization occurs due to the other product of PLC-mediated PIP2 13 hydrolytic activity, DAG, which stimulates protein kinase C, which in turn activates the Na^-

antiporter (Shen, 1989).

Both PLCp and PLCy appear capable of initiating a Ca2^ increase in several animal egg systems (Kline ef a/., 1988; Moore ef a/., 1994; Shilling ef a/., 1994; Yim ef a/., 1994,

Dupont et al., 1996), including sea urchin eggs. The microinjection of methylglyoxal bis(guanylhydrazone) (MGBG), an aminoguanide capable of stimulating endogenous protein tyrosine kinase (PTK) activity, stimulated an increase in intracellular Ca2+ that was fully blocked by PTK inhibitors and was reduced by treatment with heparin or U73122 (Shen et of., 1999). This result is in contradiction with the results obtained with PTK inhibitors, which failed to inhibit the fertilization response.

Experiments that suggest a role for PLCy during the fertilization response came from studies where unfertilized sea urchin eggs were microinjected with a glutathione-S- transferase (GST) fusion protein that contained the two tandem SH2 domains of a bovine

PLCy. Microinjection of the fusion protein blocked the Ca2^ response during fertilization

(Carroll ef a/., 1999; Shearer ef aA, 1999), suggesting that IP3 production is mediated by the

PLCy isofbrm. Microinjection of a sea urchin tandem SH2 construct did not block the Ca2* response of fertilization but did block normal cleavage (Shearer ef a/., 1999). Most of the injected eggs remained at metaphase stage, some showed abnormal division, and a small proportion showed some cytolysis. The bovine fusion protein was also used to investigate the role of PLCy in the fertilization-induced Ca2* release in starfish eggs (Carroll ef a/., 14

1997). In these eggs, injection of the fusion protein delayed the Ca2* release at fertilization

and blocked it when a high concentration of protein (- 1 mg/ml) and a low dilution of sperm

were used. A further complication is the finding that PLCy is already active in the

unfertilized sea urchin egg and its activity remains unchanged for the first two minutes

following activation by sperm (De Nadai ef a/., 1998). These data are supported by Rongish

ef aZ. (1999) who demonstrated that an active pool of PLCy is present in the unfertilized egg

with the protein being already phosphorylated on tyrosine residues before fertilization.

Therefore, the mechanism by which the GST-fusion protein is acting to inhibit an already

active PLCy is not clear. Despite the discrepancy of PLCy already being active yet capable

of having its activation blocked at fertilization is extremely interesting because such

discrepancies are not only found in sea urchin eggs, but extend to other animal eggs during

fertilization as well. Several dichotomies exist between the use of PTK inhibitors and GST-

fusion proteins. PTK inhibitors block morphologic events (Sato ef aZ., 1998) and the Ca^

wave (Glahn and Nuccitelli, 1999) in J&nqpwa eggs during fertilization, while the GST-

fusion protein did not inhibit the Ca^ rise (Runft ef a/., 1999). Runft and colleagues (1999)

also showed that an injection of an antibody against Goq, which can block its ability to

activate PLCP, does not inhibit the Ca^ response at fertilization in AeMqpws eggs. Based on

the results of this study (Runft ef a/., 1999), Ca2* release at fertilization in Aenqpwa appears

to require IP] receptors, but neither SH2 domain-mediated activation of PLCy nor Goq-

mediated activation of PLCfS. Dupont ef aZ. (1996) have demonstrated that both PLCy and

PLCP may generate IP3 to control the Ca^ response during fertilization in mouse eggs. The 15

GST-SH2 domain fusion protein did not inhibit Ca^ release during fertilization in mice

(Mehlmann ef oA, 1998).

The following experimental results suggest that the production of IP3 is mediated by

PLCp activation within the sea urchin egg. Cortical vesicle exocytosis (caused by a rise in

[Ca2^]i) can be induced in unfertilized sea urchin eggs by injecting GTPyS (Turner ef aA,

1986). Microinjection of whole cholera toxin (CTX) (which catalyzes the specific ADP-

ribosylation of G proteins (Gill, 1982)), or CTX subunit A (the subunit which catalyzes

ADP-ribosylation) is also capable of inducing exocytosis of cortical vesicles in a Ca2*-

dependent manner in unfertilized sea urchin eggs (Turner et al., 1987). Microinjection of

GTPyS into unfertilized sea urchin eggs is capable of triggering an increase in [Ca^j similar

to the fertilization response (Crossley ef aA, 1991). Pretreatment with U73122, a PLCP

inhibitor, blocked both the GTPyS as well as the fertilization responses, supporting the theory

that both signaling pathways involve PLC(3 (Lee and Shen, 1998). Pre-loading sea urchin

eggs with GDPPS did not inhibit the rise in intracellular Ca2^ during fertilization (Whitaker

gf

insemination (Crossley ef a/., 1991). This increase in Ca2^ as a result of injecting GDPpS

has been observed in other animal eggs but its mechanism is currently unknown.

JWe of ofAer fLC Afrfwg

In addition to the previously mentioned isofbrms of PLC, a novel PLC6 was isolated

from the egg of the sea urchin, Arongy/ocenfrofwa pw/pwroAzf (Coward ef a/., 2004). This

PLC was also found in the sperm and in the early embryo. The presence of the protein was 16 demonstrated in ZjfgcMnwa /%cf%s egg cytosolic fractions and sperm protein extracts using mammalian anti-PLCÔ-2 antibodies. The PLCÔ isofbrm was cloned from & pmpwrafwy egg

and developmental expression was analyzed by RT-PCR and Northern blot throughout early

embryonic development and by whole-mount in situ RNA hybridization blastula, gastrula,

and pluteus stages. Although the presence of the mRNA was demonstrated 6om unfertihzed

egg through larval development, additional work needs to be done in order to determine the

level of protein expression as well as the functional role of this PLC. The only known

mechanism for regulating PLCS activity is via Ca2\ so it is unlikely that this PLC is involved

in initiating increases in Ca2* levels, but it may be involved in propagating them once

initiated by one of the other PLC isoforms.

The most recently identified PLC, PLCÇ, appears to be specific to sperm and has been

implicated in egg activation in mammals (Saunders gf a/., 2002; Swann gf a/., 2004). In

addition to mice, where it was first discovered, PLC^ has also been identified in sperm

extracts from hamster, pig, and humans (Saunders gf a/., 2002; Swann gf a/., 2004).

Microinjection of PLCÇ into mouse eggs induces Ca2* oscillations that are indistinguishable

from those induced by fertilization as well as normal development to the blastocyst stage.

Removal of PLCÇ from sperm extracts abolishes the Ca2* release in eggs. Based on these

findings, it has been proposed that PLC^ may act as the molecular trigger that initiates the

developmental process going from fertilized egg to embryo (Swann gf af., 2004). Although it

is capable of causing Ca2^ release in sea urchin egg homogenates (Saunders gf a/., 2002), this

novel, sperm-specific PLC^ has not been identified in any non-mammalian organisms. 17

In the sea urchin egg, changes in ionic activity during fertilization are both necessary and sufficient for activation of the developmental program. An early transient increase in

[Ca^]i is critical for this process and has been extensively characterized. The egg contains both the traditional IP3 receptor and ryanodine receptor release mechanisms for intracellular

Ca2\ The natural ligand for the ryanodine receptor is thought to be cADPR, which is regulated by cGMP-mediated signaling pathway. This does not appear to be the pathway

that is involved in regulating the release of Ca2+ at fertilization based on experiments using

inhibitors of the individual components of this signaling cascade. None of the inhibitors of

the NO/cADPR signaling pathway were able to block the fertilization-induced rise in [Ca2+];.

Various inhibitors of the IP3 receptor were capable of inhibiting the fertilization-induced

increase, indicating that this is the signaling mechanism that is responsible. This is further

supported through the use of U73122, an inhibitor of the enzyme PLC that is responsible for

generating IP3, which also blocks the fertilization-induced increase in [Ca2^];. Although it

may be agreed upon that this is an IPs-mediated event, there is still some discrepancy as to

which isofbrm of PLC, PLC(3 or PLCy, is responsible for generating this signal at

fertilization.

Evidence both supporting and refuting the involvement of each isofbrm exists. Some

evidence indicates that PLCy is involved. For example, fusion protein constructs are

purported to be inhibitory, yet pharmacological protein tyrosine kinase inhibitors have no

inhibitory effects at all at fertilization. More importantly, it has been demonstrated that PLCy

is active in unfertilized eggs (De Nadai gf aA, 1998) and phosphorylated on tyrosine residues

(Rongish gf aA, 1999). Less work has been on PLCp, although there is still indirect evidence 18 of its existence in the egg. Injection of GTPyS into the egg can trigger an increase in [Ca^j, which can be blocked with the PLC|3 inhibitor U73122, just as it is blocked during fertilization. Based on the results presented in the introduction, it is clear that understanding how sperm activates the egg at fertilization, requires description of the roles of PLCy and

PLCp during this event. The focus of this dissertation to investigate the role ofPLCp.

The main focus of this dissertation research was to concentrate on the heterotrimeric

G protein-regulated isozyme of PLC and its involvement during fertilization of the sea urchin egg. Since there has been only indirect evidence for such an involvement of this enzyme through the use of pharmacological agents that are agonistic or antagonistic for heterotrimeric G proteins, isolation of the PLC enzyme itself would be immediately beneficial for two reasons. First, it would demonstrate directly that all of the components of this signaling pathway, the heterotrimeric G proteins and their effector, PLC(3, are present in the egg. Second, by identifying the isofbrm that is present in the egg, a better idea of its regulation might shed some light on the mechanism of egg activation by sperm. Once the protein was identified, specific molecular and biochemical techniques would be used to isolate it and characterize its activity m vzfro. This system would provide a more controlled environment for determining how its activity is regulated without the limitations that are inherent in working with an intact cell. Individual signaling components could be

manipulated without other cellular components being present to interfere with the specific

experimental questions that are being asked. This characterization of PLCP activation may 19 show that the isofbrm present in the sea urchin egg is activated by G^y dimers rather than

Gctq, as was found in Aemopwa eggs. These results would shed a new light on previous experimental results that manipulated what was thought to be the only signaling component,

Goq. An understanding of how the enzymatic activity of the PLC(3 from sea urchin eggs is regulated in an m system could then be utilized in egg homogenate and m vzvo studies.

It is within this environment that the physiological role of this PLCP can be determined, specifically, its involvement in the increase of calcium activity that occurs in response to fertilization. Understanding the regulation of the initiation of a brief increase of intracellular calcium at the onset of fertilization will help us understand how the sperm activates the developmental program in the sea urchin egg. 20

CHAPTERS. MATERIALS AND METHODS

ffgwwdoM ofgwwefes

Sea urchins, Zyfec&fMwa jPicfwa, were purchased from commercial suppliers and

maintained in Instant Ocean culture with biweekly fieedings of A&zcrocywfM. Eggs and sperm

were obtained by injection of 0.5 mM KC1 into the coelomic cavity. Sperm was collected

dry and stored at 4°C. Eggs were shed into artificial sea water (ASW) of the following

composition: 470 mM NaCl, 10 mM KC1, 11 mM CaClz, 29 mM MgSO#, 27 mM MgClz, 5

mM NaHCOs, pH 8. The jelly coats were removed from the eggs by several passages

through Gne-mesh silk and then allowed to settle through several ASW washes. Dejellied

eggs were then kept at 16-18°C in suspension by constant stirring at 60 rpm until used. For

insemination, sperm was diluted in ASW and added to the eggs to make a final dilution of

approximately 1:15,000.

of a «o/ènw wx fAe egg.

In order to determine the identity of PLCP within the sea urchin egg, a polymerase

chain reaction (PCR)-based strategy was utilized. Two sets of sense and antisense

oligonucleotide primers designed from known PLCP amino acid sequences were synthesized

and used to probe a cDNA library generated from sea urchin (ZjfecAznws ovarian

tissue (Rakow and Shen, 1994). These primers were selected by aligning several known

amino acid sequences of PLCp isofbrms and locating regions of evolutionary conserved

sequence homology. The following sequences were obtained from GenBank and aligned 21 using CLUSTAL software: Aernopit; /aevis PLC1, human PLC-^2, rat PLC-^1, hydra PLC- pH2, human PLC-(33, DroaopAi/a wo/p/i, hydra PLC-pHl, and sponge PLC-PS. In order to generate primers for regions specific to PLCP, amino acid sequences were also aligned for the following non-P isofbrms of PLC: rat PLC-64, human PLC-Ô1, hydra PLC-ÔH, human

PLC-yl, PLC-yD, sponge PLC-yS, human PLC-y2, and C. R05G6.8.

For the first round of PCR, primers for the amino acid sequences SVEMYRQ and

VEVDMFG (corresponding to amino acids 345-351 and 697-703, respectively, of rat brain

PLCP) were used. For the second PCR reaction, primers for the amino acid sequences

CVELDCW and FWN(A/V)GCQMV (corresponding to amino acids 359-365 and 614-622,

respectively, of rat brain PLCP) were used. The codon sequences for the amino acids used

were selected based on codon usage tabulated from 42 sea urchin (.Sfrongy/oceMfrofwa sp.)

genes in Wada ef a/. (1991) in order to reduce the amount of degeneracy within the primers.

After the first round of amplification, the products of the first PCR reaction serve as the

template for the second/nested reaction in which the primers are internal to the first set of

primers used. Utilizing this method for designing the oligonucleotide primers allows for

increased specificity in amplifying the sequence of interest (Dupont ef a/., 1996; Koyanagi ef

of., 1998).

OAfaùxwxg «wwpfgfe aegwewce of PLC/7f&e egg

Initially, the amplified fragment was used to probe the cDNA library in order to

obtain as much of a full-length clone as possible. After numerous screens of the entire

library, no other clones could be isolated. Utilizing the obtained partial sequence to generate 22 new primers, 5'- and 3'- rapid amplification of cDNA ends (RACE) reactions were performed following the manufacturer's protocol (SMART RACE cDNA Amplification Kit,

Clontech). The gene-specific oligonucleotide primers used in these experiments were cgtccgtattgcctggtggggaaacagca and gcaaagaagggggagctgttatcacaagaa for the 5'-RACE reactions the 3'-RACE reactions, respectively. The single-strand cDNA template for these reactions was generated via a reverse transcription (RT) reaction of total RNA isolated from unfertilized eggs (NucleoSpin RNA II Kit, Clontech). Total RNA was also isolated from the ovary for generating a second cDNA template. This second set of reactions was run to test if the original fragment amplified from the sea urchin ovary cDNA library is present in just the egg.

GenerodoM of agaùisf sea «rcAwx PLC/? (SwPLC/^

Based on the sequence data obtained from the RACE reactions, an antibody was generated against a specific epitope determined from the translated sequence of the sea urchin enzyme. A specific region of the protein comprised of fourteen amino acids

(SGSSIAAEDEIKLK) that are located in the "hinge region" of the enzyme between the X and Y domains was selected as the epitope for generating a rabbit polyclonal antibody. This epitope sequence was selected for two reasons. First, it appears to be an amino acid sequence that is unique to the sea urchin PLCp sequence as compared with other known

PLCP sequences, thereby increasing the specificity of the antibody. Second, the hinge region is in between the X and Y domains of the enzyme, the catalytic core of the enzyme that

forms a binding pocket for the inositol head group of PIP2, and ultimately hydrolyzing it to

generate IP3. This region has not been studied in any great detail, but is thought to play an 23 important role in the enzymatic activity of PLC. The peptide was synthesized and conjugated via an additional cysteine residue added to the end of the peptide, to keyhole limpet hemocyanin (KLH). The peptide-KLH conjugate was then injected into the host rabbits with additional boosters and serum bleeds as per the company's (Sigma-Genosys) protocol. A portion of the final bleed was further affinity purified using the peptide immobilized on sepharose beads as per the company's protocols.

Dejellied eggs were spun down in a hand-centrifuge and the ASW was aspirated off.

An equal volume of ice-cold RIPA buffer (10 mM Tris pH 7.0 for unfertilized eggs or pH 7.5 for fertilized eggs, 150 mM NaCl, 200 pM PMSF, 200 pM NagVO^, 50 mM NaF) without detergents was added to the volume of packed eggs yielding a 50% (voUvol.) suspension.

The eggs were lysed by shearing with a narrow gauge needle and syringe. The lysate was centrifuged at 100,000xg for 30 minutes at 4°C and the supernatant was saved as the soluble protein fraction. The remaining pellet was resuspended with ice-cold RIPA buffer of the appropriate pH containing detergents (1% NP-40, 1% deoxycholic acid, 0.1% SDS), and incubated on ice for 15 minutes. It was then centrifuged at 100,000xg for 30 minutes at 4°C and the supernatant was saved as the particulate protein fraction. The total protein concentration in the lysates was quantified using the DC-Protein Assay reagents (BIO-RAD).

Equal concentrations of total protein were loaded for SDS-PAGE on various percentage gels

(Laemmli, 1970). Protein was transferred to nitrocellulose membranes after equilibration in transfer buffer (Towbin ef oA, 1979) using BIO-RAD's Semi-Dry Transfer apparatus. After transfer, the membrane was blocked for 1 hour at room temperature with TBS-T (Tris 24 buffered saline, 0.5% Tween-20) + 5% milk, washed 3 times with TBS-T, and then incubated overnight at 4°C with the primary antibody diluted in TBS-T +5% BSA. The following day the membrane was washed 3 times with TBS-T, and then incubated for 1 hour at room temperature with the secondary antibody (conjugated to HRP) diluted in TBS-T +5% milk.

After a final 3 washes with TBS-T, the immunoreactive bands on the membrane were visualized using chemiluminescence reagents (Pierce Biotech., Inc.) on x-ray 61m.

Geaeref&w; of a/WWgwgfA cfoae of

In order to generate a clone with the complete coding sequence for the protein from the three individual amplified products, a PCR-based strategy was utilized. A primer for the

5' end start region (caccatggctaagcaatatg) was generated and the 5'-RACE product and the original fragment cloned from the cDNA library served as the templates for the PCR reaction. The newly generated 5'-end coding fragment, along with the 3'-RACE product were used as templates for another PCR reaction with a new 3'-end primer

(tttttgctaatgttcctgttg) designed to be used with the 5'-end start primer. A proof-reading DNA polymerase was used in all reactions to minimize errors. The full-length clone was ligated into the TOPO-pCR2.1 expression vector following the company's protocol (Invitrogen) with subsequent transformation into DH5a cells. Plasmid was isolated from the cells grown in culture using Qiagen's Miniprep kit, and the sequence was verified by restriction enzyme digest analysis, nucleotide sequencing of the 5' and 3' ends, and expression of the ^S-labeled protein in a rabbit reticulocyte lysate transcription/translation system (Promega). 25 jkcwmMngaf agprewfow /Mfrf/kadow

The full-length clone in the TOPO-pCR2.1 vector was used as a template for PCR reactions to generate Ndel (ccgcgcatatgcaatggctaagcaatatg) and NotI (tagaaggcacagtcgagg) restriction sites in the product for ligation into the pAcGHLT-A Baculovirus transfer vector

(BD Pharmingen). Expression results in a fusion protein with a GST tag followed by a 6xHis tag at the amino terminal end. The ligated product was transformed into TOPIO cells

(Invitrogen) which were then used to inoculate a larger-scale culture to generate a sufficient amount of the vector for transfection reactions. The vector was purified from the cultures using Qiagen's MIDl-Prep Kit.

The day before the transfection, a 6-well plate was seeded with 4x105 SJ9 cells per well. For the transfection reaction, 0.1 pg of Baculogold DNA (BD Pharmingen) was mixed with 0.4 pg of the purified transfer vector and the final volume was brought up to 100 pi with

Buffer EC to which was added 3.2 pi of Enhancer (Effectene Transfection Reagents from

Qiagen). The solution was briefly mixed and incubated for 5 minutes. Next was added 10 pi of Effectene, vortexed briefly, and incubated for 10 minutes. Next, 600 pi of TNM-FH

Insect medium (BD Pharmingen) was added, pipetted up and down several times, and then added drop-wise to the well containing the ,5/9 cells plated the previous day. The plate was swirled gently to mix the reagents, and incubated at 27°C for 7 days. Upon verification that the cells were infected with the recombinant Baculovirus, the media was removed and added to a 25 ml tissue culture flask (T25) containing cells at >85% confluency. These cells were then incubated at 27°C for another 7 days. The media was then removed and used to infect a 75 ml flask (T75) containing 6/P cells at >85% confluency. After incubating at 27°C 26 for 4 days, the viral titer in the media was high enough to infect subsequent T75 cultures at 1 ml per flask.

For generation of recombinant protein, 10 T75 flasks at >85% confluency of 5/9 cells were infected with 1 ml of viral media per flask. The flasks were incubated at 27°C for four days, at which point the cells were harvested by centrifugation at lOOOxg at 4°C. To the cell pellet was added 20 ml of ice-cold modified RIPA buffer (10 mM Tris pH 7.5, 150 mM

NaCl, 200 pM PMSF, 200 pM NasVC^, 50 mM NaF, 1% NP-40,1% deoxycholic acid, 0.1%

SDS, 40 mM myo-inositol, 2 mM diCTPC (l^-diheptanoyl-fm-glycero-3-phosphocholine),

100 pM CaClz) and the pellet was disrupted using a narrow gauge needle on a syringe. The lysate was incubated on ice for 30 minutes and then centrifuged at 21,700xg for 30 minutes at 4°C. The supernatant was transferred to a new tube, 300 pi of a 50% slurry of Glutathione

Sepharose 4B (Amersham Biosciences) was added, and the solution was incubated rocking for 2 hours at 4°C. The solution was then centrifuged at 500xg for 5 minutes at 4°C, the supernatant was removed, and the beads were washed 3 times with the modiSed RIPA buffer. After the final wash, 150 pi of modified RIPA buffer was added resulting in a 50% slurry of recombinant GST-suPLCp on the Glutathione Sepharose beads to be used for subsequent activity assays.

Alternatively, the recombinant suPLCp was also removed from the beads via proteolysis. After the final wash as mentioned above, the GST-suPLCp on the Glutathione

Sepharose beads was resuspended in 1 ml of PBS containing 5 0U of Thrombin and incubated on a rocker for 16 hours at 22°C. The proteolysis reaction was centrifuged at lOOOxg for 3 min. at 4°C and the supernatant was transferred to a new tube. In order to remove the 27

Thrombin, the supernatant was passed through a HiTrap Benzamidine FF (high sub) 1 ml column following the manufacturer's protocol (Amersham Biosciences). Following loading of the supernatant on the column, the Grst three 1 ml elution fractions were collected and analyzed by SDS-PAGE followed by silver staining of the gel and by Western blot analysis with the anti-suPLCp antibodies. Eluted fractions were then used for subsequent activity assays.

PLC gwgwHzdc acdwfy assay

The PIPz-specific hydrolytic PLC activity of the recombinant protein was quantified using a modified protocol of the one used for PLCy from sea urchin eggs (Rongish et al,,

1999). PLC activity was measured in 43.5 pi of PLC buffer (50 mM NaF^PC^, 100 mM

KC1, 1 mM EGTA, pH 6.8, 2 mM diCTPC) containing 6 nmol of a mixture of PIP; and

[3H]PIP2 (5.6 pCi/pmol) in a mixed micelle formed by 0.12% octyl-glucopyranoside +

0.02% deoxycholate. CaClz is added to a final concentration to give the desired free calcium concentration for a given reaction. To this reaction buffer 5 pi of the purified 50% slurry as well as any other activators, inhibitors, etc. were added. The reactions were carried out for

60 minutes at 37°C with agitation, and stopped by addition of 100 pi of 1% BSA followed by

200 pi of 10% TCA. The reactions were then centrifuged at 26,000xg for 5 minutes and 200 pi of the TCA-soluble material (IP3, the product of PIP; hydrolysis) was counted in a scintillation counter. 28

Just as in other cell systems (Wahl ef o/., 1992), the PLOy activity in the egg is absolutely dependent on the concentration of free Ca^ (Rongish ef a/., 1999). Since the binding of a Ca^ in the catalytic domain appears to be a requirement of PLC enzymes (Ellis ef o/., 1998), the calcium dependence of the PLCp in this study was also investigated. The enzymatic activity was assayed under buffering conditions designed using the Maxchelator software package (Patton ef oA, 2004; http://www.stanfbrd.edu/-cpatton/maxc.htmll to provide deGned levels of 6ee calcium in order to determine the calcium dependence of suPLCp.

Higferofnmenc G /wvfewi gxyrgssfow

Baculovirus constructs for Goq, GPi, and Gyz-His were generously provided by S.

Scarlata (SUNY, Stonybrook). Expression and purification of these subunits was carried out

based on the protocol of Kozasa and Gilman (1995) with some modifications. A 2 liter

suspension culture of cells were grown in TNM-FH insect medium (BD Pharmingen)

supplemented with 1% Pluronic F-68 and 1% lipid mix (Life Technologies, Inc.). The

culture (-1.5 x 10^ cells/ml) was infected with amplified recombinant Baculoviruses

encoding each of the G protein subunits at a ratio of 1:1:1. Cells were harvested 48 hours

later by centrifugation at lOOOxg for 10 minutes and then resuspended in 120 ml of lysis

buffer (50 mM Hepes pH 8.0, 0.1 mM EDTA, 3 mM MgCl2,10 mM P-mercaptoethanol, 100

mM NaCl, 50 pM GDP, and protease inhibitors). Remaining procedures were carried out at

4°C unless otherwise specified. The cell lysate was centrifuged at 100,000xg for 30 minutes 29 and the resulting pellet was resuspended in 120 ml of wash buffer (50 mM Hepes pH 8.0, 3 mM MgClz, 10 mM (3-mercaptoethanol, 50 mM NaCl, 50 pM GDP, and protease inhibitors), homogenized using a Dounce homogenizer, and centrifuged at 100,000xg for 30 minutes.

The pellet, resuspended in 80 ml of wash buffer, was frozen in liquid nitrogen and stored at

-80°C.

The frozen sample was thawed in room temperature water, diluted with 60 ml of wash buffer containing 1% (w/v) sodium cholate, stirred on ice for 1 hour, and then centrifuged at 100,000xg for 40 minutes. The supernatant (membrane extract) was diluted 4x with buffer A (40 mM Hepes pH 8.0, 2 mM MgClz, 2 mM P-mercaptoethanol, 200 mM

NaCl, 50 pM GDP) and loaded onto a 5 ml Ni-NTA (Qiagen) column that had been equilibrated with 8 column volumes of buffer A. The column was washed with 5 column volumes of buffer A containing 300 mM NaCl and 5 mM imidazole. The column was then incubated at room temperature with buffer F (20 mM Hepes pH 8.0, 0.2 mM MgClz, 10 mM

P-mercaptoethanol, 100 mM NaCl, 5 pM GTPyS, 0.2% sodium cholate, and 5 mM

imidazole) and washed with 32 ml of the same buffer. Goq was eluted at room temperature

with 10 ml of buffer E (20 mM Hepes pH 8.0, 10 mM P-mercaptoethanol, 50 mM NaCl, 10

pM GDP, 1% sodium cholate, 30 mM AICI3, 50 mM MgClz, 10 mM NaF, and 5 mM

imidazole) followed by elution of G^y-His with high imidazole. Fractions were analyzed by

Coomassie staining and Western blot. Positive fractions were pooled and dialyzed with

buffer D (20 mM Hepes pH 8.0, 1 mM EDTA, 3 mM MgClz, 3 mM DTT, 0.7% CHAPS)

followed by addition of 10% glycerol, freezing in liquid nitrogen and storing at -80°C. 30

In order to activate the purified Goq subunit, the purified protein was added to an activating buffer (6 mM MgClz, 20 pM AICI3, 10 mM NaF, 1 mM EDTA, 300 pM GDP) to a final reaction volume of 200 pi. The reaction was incubated at 30°C for 1 hour with gentle rocking at which point it was added to subsequent activity assays at 30 pi per assay. 31

CHAPTER 4. RESULTS

gfwf cfoamg of a «o/ônw Ae egg

Previous experimental results suggest that there is a G protein-regulated isofbrm of

PLC present within the sea urchin egg. The evidence that strongly supports this is the ability of GTPyS to trigger a Ca^ response that is similar to the fertilization-induced response, and that it is blocked when the eggs are pretreated with the PLC(3 inhibitor, U73122 (lee and

Shen, 1998). Initial studies of egg protein lysates using commercially available antibodies against the various isoforms of PLCp were unsuccessful All of the antibodies were generated against epitopes present in the mammalian isoforms of the enzymes which could be different or completely absent in the sea urchin isofbrm. In order to identify the isofbrm in sea urchin eggs, a PCR-based strategy was utilized.

Degenerate-nested oligonucleotide primers designed from known PLCP amino acid sequences were used to probe a cDNA library generated from sea urchin (lyfccAmwa pzcfzw) ovarian tissue. By means of agarose gel electrophoresis, a single PCR product was isolated

(Figure 1A.), purified and subcloned into a vector. The clone was sent to MWG Biotech for sequence determination. The resulting nucleic acid sequence of the PCR product was translated, and by comparing the partial sea urchin amino acid sequences against known

PLC|3 sequences from other organisms (Figure 2), it was confirmed that a PLCp isozyme was indeed cloned from the sea urchin cDNA library. Based on this partial cDNA sequence,

it was not possible to determine which of the four known PLC(3 isoforms the sea urchin egg

sequence was most homologous to without a complete coding sequence. 32

Initially, the amplified fragment was used to probe the cDNA library in order to obtain as much of a full-length clone as possible. After numerous screens of the entire library, no other clones could be isolated. Utilizing the obtained partial sequence to generate new oligonucleotide primers, 5'- and 3' RACE reactions were performed on single-strand cDNA templates generated via RT-PCR reactions of total RNA isolated from sea urchin ovaries. Total RNA was also isolated from unfertilized eggs to generate a second cDNA template in order to verify that the original fragment amplified from the sea urchin ovary cDNA library is present in just the egg. As seen in Figure IB, both the 5'- and 3'-RACE reactions each yielded a single product in both the ovarian and egg samples, confirming that the sequence is present within the egg itself. The egg RACE reaction products were purified and then sent to MWG Biotech for sequencing. The nucleic acid sequences for the 5'- and

3'-RACE reaction products were then assembled with the original fragment in order to generate the full-length coding sequence.

The complete coding sequence is 3468 base pairs in length, yielding a translated product of 1148 amino acids with a predicted molecular mass of 132 kDa and a pi of 6.47

(Genbank accession #AY550251). Sequence analysis indicated that the translated protein contains the characteristic domains that are found in other known PLCP sequences (Figure

3). The catalytic core (X and Y domains) is flanked by a PH and EF hand domains upstream of the X domain at the amino-terminal end, and by a C2 domain followed by a long carboxy- terminal sequence downstream of the Y domain. Based on these similarities in domain organization with known PLC|3 sequences, the cloned sequence subsequently will be referred to as suPLCp (sea urchin PLCp). 33

Compar&oM segifewce w^A Anown fLC^ segwewcef

There are four isoforms present within the PLCP subfamily referred to as PLCpi-4

(Rebecchi and Pentyala, 2001; Rhee, 2001). In order to determine to which isoforms the suPLCp is most similar, the amino acid sequence of suPLCP was aligned individually with each of twenty amino acid sequences of different PLCp isoforms from eleven different species using ClustalW sequence alignment (Appendix). Based on these alignments, suPLCp shares the highest degree of similarity with rat PLC(34 (63% identity, 79% homology) followed closely by mouse PLC(34 (62% identity, 79% homology) and the least with sponge PLCP (34% identity, 47% homology), closely followed by both human and rat

PLC(32 (35% identity, 58% homology in both cases) (Figure 4). Based on these alignments, it was concluded that suPLCp shared the greatest degree of identity with the mammalian

PLCP4 isofbrm, an interesting finding since expression of PLCP4 is limited to the retina and certain neuronal cells while PLCpi and PLCp3 are expressed more widely.

GkMena&m of a /kfWeMgf* c&me of

In order to assemble the three individual cloned fragments of suPLCp into a single clone containing the entire coding sequence, a PCR-based strategy was utilized. As mentioned in the Materials and Methods, new oligonucleotide primers were synthesized to first combine the 5'-RACE fragment with the original cloned fragment, the product of which would then be used in a reaction to combine with the remaining 3'-RACE fragment, thus generating a single full-length clone. The resulting 3.4 kb clone was ligated into a pcDNA3.1 expression vector (Invitrogen) which was verified by partial sequencing and 34 restriction digest analysis. The expression vector containing the full-length clone was then added to rabbit reticulocyte lysate mix with ^S-methione in order to verify translation of a protein of the predicted molecular mass. As seen in Figure 5, a single ^S-labeled protein with a calculated molecular mass of -132 kDa is present in the lanes loaded with lysate from the reaction that had the vector with the 3.4 kb insert present versus those lanes loaded with lysate from the reaction that contained the vector without the insert present.

flreseace ofswPLC/?w&Aùx dke egg

Once the nucleic acid sequence was isolated from the egg, the next step was to verify the presence of the protein within the egg. Since none of the commercially available anti-PLCp antibodies exhibited any immunoreactivity with protein lysates generated from sea urchin eggs, a rabbit polyclonal antibody was generated recognizing a specific epitope determined from the translated sequence of the sea urchin enzyme. The epitope region which was selected is located between the X and Y domains of the protein and appears to be a unique sequence to suPLCp as compared with other PLC(3 sequences. Both soluble and particulate protein lysates were prepared from unfertilized eggs and eggs one minute post- fertilization. The protein concentration was quantified for each of the lysates and equal amounts of total protein were loaded for SDS-PAGE, transferred to a nitrocellulose membrane, incubated with the anti-suPLCp antibodies, then with HRP-conjugated anti-rabbit

Ig antibodies, followed by visualization of the immunoreactive bands via chemiluminescence on X-ray film. As seen in Figure 6A, immunoreactive bands of the predicted molecular mass are present in both the unfertilized and one minute post-fertilization lysates, specifically, in

the particulate fractions. There were no bands of similar molecular mass present in either of 35 the soluble lysates or in a lysate of sea urchin sperm. In order to verify the specificity of the anti-suPLCp for the immunoreactive bands of 132 kDa, as well as for the other smaller immunoreactive proteins present on the blot, the Western was repeated. One blot was treated as before and a second blot had the anti-suPLCp antibody pre-incubated with the epitope peptide in order to block the antigen-specific binding site. The 132 kDa bands were no longer present in the blot where the primary antibody was blocked with the peptide indicating a specific antibody-antigen interaction, whereas the other bands present were most likely due to non-specific interactions with either the primary or secondary antibodies (Figure 6B).

Presence of swfLC/? fn of&er specks of sea wrc&wx

In order to determine how conserved the suPLCp protein is, its presence was tested for in two species of sea urchin other than Zyfec&zMws /Hcfws, in which it was identified. In the first species, Z,)#ecAmws varzegafzw, protein lysates were generated from both unfertilized and fertilized eggs as for Z,. pzcfws. As seen in Figure 7, when the lysates were probed with the anti-suPLCp antibody, an immunoreactive band was present in both the unfertihzed and fertilized particulate fractions indicating that a similar protein is present in two different species within the same genus. The specificity of this reaction is demonstrated when the primary antibody is once again pre-incubated with the blocking peptide and the 132 kDa bands are no longer present in the Western blot.

The second species of sea urchin used was .RroMgy/ocemfrofMs pw/pwrafwa, whose genome is in the process of being sequenced (Cameron ef a/., 2000). The sequence information that is being collected is available on-line (http://su2D.caltech.edu/) although the annotation of the sequences has not yet been completed. The 1. jwcfws amino acid sequence 36 was used in a BLAST search of the complete & pwfpwrofws database in order to identify any similar sequences from this species of sea urchin. Figure 8 shows the complete Z. p;cfwa suPLCp sequence with corresponding sequences identified from the & /w/ywroAt; database indicated. Based on the unassembled comparison of the & sequence, there appears to be a sequence that has a high degree of identity to suPLCp present in this species of sea urchin as well.

EayreM&wx endof recomMagMf

As stated earlier, suPLCp protein was detected in protein lysates from sea urchin eggs via Western blot analysis using the anti-suPLCp antibody. Unfortunately, this antibody was not able to immunoprecipitate a sufficient quantity of the protein from the lysates for further characterization of its enzymatic activity. To further analyze its activity, a recombinant suPLCp was generated that, when expressed, resulted in a fusion protein with a

GST tag at the amino terminal end. This fusion tag was used for purification of the

expressed protein. Initially, expression of the fusion protein was attempted in a bacterial

expression system. Several bacterial strains were tested under various expression conditions

with the same result in each case, the expressed protein would be in inclusion bodies within

the bacteria. Several protocols were tested in order to purify the protein from the inclusion

bodies, but all resulted in extremely low recovery with no enzymatic activity. In order to

overcome the problems of expressing a large fusion protein (-160 kDa) that appeared

insoluble in a bacterial expression system, another protein expression system was utilized. 37

The full-length clone coding for suPLCp was used in a Baculovirus expression vector system. The main reason for choosing this system is that it can handle higher levels of large protein expression than prokaryote systems. It typically produces a protein that is similar to its native counterpart, both structurally and functionally, and once established, it is a relatively easy system to use. For expression, the full-length suPLCp clone was ligated into the pAcGHLT-A Baculovirus transfer vector (BD Pharmingen), once viral titers reached sufficient levels, they were used to infect Sf9 insect cells (established from ovarian tissues of

Spodoptera frugiperda larvae). Expression results in a fusion protein with a GST tag followed by a 6xHis tag at the amino terminal end, with the GST tag being used for subsequent purification of the recombinant protein.

After infection with the recombinant Baculovirus, the infected cells were harvested as outlined in the Materials and Methods. Aliquots were saved from each step of the initial procedure in order to verify the presence of the recombinant fusion protein by Western blot analysis using the anti-suPLCp antibody. As seen in Figure 9, there is an immunoreactive protein present in the initial lysate of the predicted molecular mass (-160 kDa). This protein is then purified from the total cell lysate using Glutathione Sepharose 4B beads, but even in

the presence of excess Glutathione (20 mM), it was not possible to elute the protein off the

beads. This was unexpected, so enzymatic activity assays were run using the protein bound

to the sepharose beads (Figure 9). The enzymatic assays from these initial purifications

indicated that the recombinant protein did not possess any PIP; hydrolytic activity. Upon

reviewing the cell lysis and protein purification conditions, modification to the composition

of the solutions was made in order to maintain the protein conformation through the

purification process. 38

Initially, the traditional formulation for RIPA buffer was used to lyse the infected

,SyP cells. The recombinant protein that was bound to the Glutathione Sepharose beads was washed and then resuspended in PBS (phosphate-buffered saline), pH 7.5. In an attempt to maintain the structural conformation of the recombinant protein, three components were added to the RIPA buffer. The first two were myo-inositol and Ca^% in the form of CaClz.

We attempted to achieve stabilization of the structural confirmation of the recombinant protein by adding a source of Ca^ (CaCl%) as well as a pseudo-substrate (myo-inositol) to occupy the catalytic site. Crystallographic studies of complexes of PLCô-1 with Ca^ and

Ins(l,4,5)Ps revealed complex interactions with the catalytic Ca^, involving several negatively charged residues in the active site of the enzyme (Essen et al., 1996; Katan, 1998).

It has been accepted that the other isoforms of PLC behave in a similar manner and require calcium for catalytic function. The coordination of Ca2+ in two other domains, the EF-hand and C2 domains has also been proposed to play a potential structural role as well (Essen et a/., 1996).

The third component that was added to the RIPA buffer was the short-chain phospholipid, diheptanoyl-PC (diCTPC) that acts as an interface for the recombinant protein.

A common property of water-soluble phospholipases is "interfacial activation", the preference of aggregated over monomeric substrate. Interfacial activation is thought to involve changes in enzyme conformation, presumably caused by the surface interacting with the enzyme at a site that is distinct from the active site. The surface binding switches the enzyme to a more active state, and therefore represents allosteric regulation. This regulation of activation exists in mammalian PLCÔ-1 (Rebecchi ef a/., 1993) and in the phosphatidylinositol-specifc phospholipase C (PI-PLC) from fAwrmgie/ww where 39 the presence of detergents profoundly increased the enzyme efficiency, affecting both turnover number and, in the case of diCTPC, substrate affinity (Zhou ef of., 1997). The presence of these three additional components in the "modified" RIPA buffer did not alter the yield of purified recombinant suPLCp or its inability to be eluted off of the Glutathione

Sepharose beads. However, these modifications preserved the enzymatic activity of the purified recombinant protein.

jdcfnWow of «(PLC/) emgwMgdc

The enzymatic activity of suPLCp was measured using the substrate mixture of radioisotope-labeled and unlabeled PI(4,5)Pz. Hydrolysis of the phospholipid substrate generated a soluble ^H-IP] which can be quantified by placing the soluble fraction of the reaction into a scintillation counter. As shown in Figure 10, the recombinant suPLCp alone has a low level of baseline hydrolytic activity. It is known that the PLCp isozymes are activated by G protein a and/or Py subunits (Smrcka and Stemweis, 1993; Boyer ef a/., 1994;

Katan, 1998; Rhee, 2001). The addition of purified, recombinant GPy dimer to the reaction increased the hydrolytic activity of suPLCp in a dose-dependent manner, reaching maximum activity at a concentration of 2 pM. Experiments using a crudely purified Goq protein were also performed in order to test its potential role as an activator of suPLCp enzymatic activity.

Currently, technical limitations with expression and purification of this protein, the results obtained with the given preparation were inconclusive as to the role of G

Given a means to activate the enzymatic activity of suPLCp, the calcium- dependence of that activity could next be investigated. All PLCs require Ca^ for catalytic function; the PLCÔ isozymes are most sensitive to this cation (Allen ef a/., 1997). Structural and mutational studies have identified residues within the catalytic domain that contribute to substrate recognition, Ca^ binding, and catalysis (Ellis ef a/., 1998). Activity assays were performed in which 1 pM G^y dimer was added to increase enzymatic activity of suPLCp in the presence of determined concentrations of free-Ca^ to determine if there was any effect on the activity. The concentrations of free-Ca2+ was within the physiological range that is present within the egg, 100 nM to 10 pM. As seen in Figure 11, in the presence of activating levels of GPy, there was no significant increase in enzymatic activity until the free-Ca2* concentration reached 1 pM, suggesting that as with other PLC enzymes, suPLCp also

possesses an enzymatic activity that is dependent on calcium.

jprofeo&mk of

Based on information suggesting that Goq protein has an affinity for Glutathione-

Sepharose beads (S. Scarlata, personal communication), another approach was attempted to

remove the suPLCp &om the Glutathione-Sepharose beads used in its purification. Within

the Baculovirus vector used for expressing the recombinant fusion protein in the cells

there is a Thrombin cleavage site between the GST tag and the suPLCp. After purifying the

GST-suPLCp from the total cell lysate using the Glutathione-Sepharose beads, the beads

were then incubated with Thrombin, which was then removed by passing the reaction 41 mixture through a Benzamidme column to prevent random proteolysis. To verify the presence of suPLCp, fractions were saved for SDS-PAGE analysis. Both silver staining of the gel and anti-suPLCp Western blot analyses were performed (Figure 12). In both cases, a single band was visible in the post-Thrombin treatment eluted fractions. The molecular mass of this band was smaller than the predicted 132 kDa, because suPLCp also has an internal

Thrombin cleavage site present 93 amino acids from the amino terminus (Figure 3) and does not cleave any of the known PLC domains. Proteolysis at this site yields a protein with a molecular mass of 121 kDa, the mass calculated for the band visible in the gel and on the blot.

Now that the recombinant suPLCp was no longer attached to the Glutathione beads, its activity was assayed again to see if deleting the first 93 amino acids had any effect on its enzymatic activity. As seen in Figure 13, the truncated protein retained its activity in response to increasing concentrations of G(ly dimer, except that at a concentration of 4 pM

G(3y, an inhibition of hydrolytic activity occurred. This inhibition was not observed in the

experiments with the suPLCp still attached to the beads. It may have been the result of

inaccessibility of the G protein dimer to all of the recombinant suPLCp present on the beads

due to steric hinderance and/or competition with other proteins that were also present as a

result of the purification from the total cell lysate. By removing the suPLCp from the beads,

these other factors may have been removed, thus revealing the inhibitory response. Although 42 the truncated protein responded to GPy, experiments with the available Goq were once again inconclusive.

The calcium-dependence of the truncated protein was tested under the same conditions used with the protein bound to the beads. The resulting curve indicates that the suPLCp which is missing the first 93 amino acids retains the requirement of free-Ca2+ for enzymatic activity. The required Ca^ levels are slightly lower when compared to the recombinant protein bound to the beads (Figure 14). Based on these results and the Gpy- mediated activation, it was concluded that the truncated protein was indeed the same protein that was present on the Glutathione-Sepharose beads used in the previous experiments.

In addition to the PH domain, a second potential site for GPy-interaction is a span of amino acids present in PLCP2 (Kuang ef a/., 1996). In addition to mapping out the specific region capable of binding purified Gpy, they also showed that the GST-fusion peptide of this region is capable of specifically inhibiting the GPy-mediated activation of

PLCP2. Figure 15 shows an alignment of those residues firom PLCP2 with the residues present in suPLCp. There does appear to be a high degree of identity between the two sequences over the 62 amino acids that span the binding site, with 66% (41/62) shared identity and 77% (48/62) shared homology. 43

A.

B.

figwre J. PCR products of PLCp from sea urchin. Samples were electrophoresed through a 1% agarose-TAE gel and visualized using ethidium bromide with an ultraviolet light. (A) Degenerate-nested PCR product 6om sea urchin ovarian cDNA library. Three separate reactions all yield 1 kb product. (B) 5'- and 3' RACE reaction products. The 5'- and 3'-RACE products are -2.5 kb and - 1.5 kb, respectively. Lane 1: ovarian 5'-RACE, lane 2: ovarian 3'-RACE, lane 3: egg 5'-RACE, lane 4: egg 3'-RACE. 44

Rat PLCp4 NAPVFLDRLELYQEMDHPLAHYFISSSHNTYLTGRQFGGKSSVEMYRQVL

Bovine PLCP NAPVFLDRLELYQEMDHPLAHYFISSSHNTYLTGRQFGGKSSVEMYRQVL

Human PLC(34 NAPVFLDRLELYQEMDHPLAHYFISSSHNTYLTGRQFGGKSSVEMYRQVL

Partial Sea Urchin PLC[? WIS

Rat PLC(Î4 LAGCRCVELDCWDGKGEDQE P11THGKAMCTDILFKDVIQAIKETAFVTS

Bovine PLCP LAGCRCVELDCWDGKGEDQEPIITHGKAMCTDILFKDVIQAIKETAFVTS

Human PLCp4 LAGCRCVELDCWDGKGEDQEPIITHGKAHCTDILFKDVIQAIKETAFVTS

Partial Sea Urchin PLCP AE FALCVELDCWDGRSEDQE P11THGLAMCTDIQFRDVIQAIRDTAFVTS

*********. # ********** ****** *.******. .******

Rat_ PLCP4 EYPVILSFENHCSKYQQYQMSKYCEDLFGDLLLKQALESHPLEPGRLLPS

Bovine PLCP EYPVILSFENHCSKYQQYKMSKYCEDLFGDLLLKQALESHPLEPGRPLPS

Human PLCp4 EYPVILSFENHCSKYQQYKMSKYCEDLFGDLLLKQALESHPLEPGRALPS

Partial Sea Urchin PLCp DFPVILSFENHCSKPQQLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPS

j .************ ** ;; ; ;***. ;. * . •** > ; *. ** * . ** *

Rat PLCP4 PNDLKRKILIKNKRLKPEVEKKQLEALKSMMEAGESAAPASILEDDNEEE

Bovine PLCP PNDLKRKILIKKQTTETEVEKKQLEALKSMMEAGESAAPVNMLEDDNEEE

Human PLCp4 PNDLKRKILIKNKRLKPEVEKKQLEALRSMMEAGESASPANILEDDNEEE

Partial Sea Urchin PLCP PNKLKRKILIKNKRLKPEVEQRQLELMRLRPELLEVNQEDEVEECESGYS

**,******** ; ; ..***..*** .. * * .: * :.

Rat PLCP4 IESAAD- QEEEAHPE YKFGNELSADDFS HKEAVANSV

Bovine PLCp IESAE- -QEEEAHPEYKYGNELSADDLG HKEAIANSV

Human PLCP4 IESAD- -QEEEAHPEFKFGNELSADDLG HKEAVANSV

Partial Sea Urchin PLCP DGAVSPPGNTDAHPKFKFPSKDSIGSISGSSIAAEDEIKLKHNMSVKMNA

:.::**** :::

Rat PLCp4 KKG-LVTVEDEQAWMASYKYVGATTNIHPYLSTMINYAQPVKFQGFHVAE

Bovine PLCP KKG-LVTVEDEQAWMASYKYVGATTNIHPYLSTMINYAQPVKFQGFHVAE

Human PLCP4 KKG-LVTVEDEQAWMASYKYVGATTNIHPYLSTMINYAQPVKFQGFHVAE

Partial Sea Urchin PLCP KKGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNYAQPVKFQGFDVAE *** *; ; *** * ;*,*;*.******* _ ** ;; *********** _ ***

Rat PLCP4 ERNIHYNMSSFNESVGLGYLKTHAIEFVNYNKRQMSRIYPKGGRVDSSNY

Bovine PLCP ERNIHYNMSSFNESVGLGYLKTHAIEFVNYNKRQMSRIYPKGGRVDSSNY

Human PLCP4 ERNIHYNMSSFNESVGLGYLKTHAIEFVNYNKRQMSRIYPKGGRVDSSNY

Partial Sea Urchin PLCp ERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSSNY ** * • *• *************. * * . ****************. * * *****

Rat PLCP4 MPQIFWNAGCQMVSLNYQTPDLAMQLNQGKFEYNGSCGYLLKPDFMRRPD

Bovine PLCP MPQIFWNSGCQMVSLNYQTPDLAMQLNQGKFEYNGSCGYLLKPDFMRRPD

Human PLCP4 MPQIFWNAGCQMVSLNYQTPDLAMQLNQGKFEYNGSCGYLLKPDFMRRPD

Partial Sea Urchin PLCP MPQIFWNAGCQMKGEFQHTGXRY *******.**** .*

figure 2 Amino acid alignment of partial sea urchin PLCP with known PLCP sequences. The translated sequence derived 6om the degenerate-nested PCR product aligned with the partial sequence of known PLC^s that were identified as being most homologous to the sea urchin sequence by performing a BLAST search of GenBank. An * denotes conserved amino acid identity, while a : denotes amino acid homology in that position in all four sequences. 45

MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEGREGQVLE * LSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMHMVASAPEI PE Domain AQVWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTRTFASGKTEKIVF

KSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDIGRNETEGLSVEKLIEF gJF-hand LNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSSDGLCRYLMSDENAPVFLD

RLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQVIjLAGCRCVELDCWDGRSEDO

EPIITHGLAMCTDIOFRDVIQAIRDTAFVTSDFPVILSFENHCSKPQOLKLARYCEEVLGEF X DQMATN LLTEKLDDHPLDASVPLPSPNKLKRKILIKNKRLKPEVEQRQLELMRLRPELLEVNQEDEVE

ECESGYSDGAVS PPGNTDAHPKFKFPSKDSIGSISGSSlAAEDEIKLKHNMSVKMNAKKGEL

LSQEDEAAWAQYRYTGATTNIHACLSRLVNYAQPVKFQGFDVAEERNLHF3SIMSSFNESVGL

GYIKTQCIEFVNYNKRQMSRIYPQGRRYDSSNYMPQIFWNAGCQMVAIjNFQSPDIGMOLNOG Y DQMATff KFEYNGCCGYLLKPDFMRRPDRTFDPFSESPVDGVIAATCSVQVISGQFLSDKKVGTYVEVD

MYGLPTDTIRREHRTRTVPNNGLNPVYNEEPFVFRKWLPDLAVLRIAVYEEGGKLIGQRIL C2 DQMATfT PLDGLQAGYRHISLRTEGNFPLSLPTVFCCIQLKTYVPDGLTQFVDALSDPQLYLSQAEKRA KQLLCMGIEENEISEVPTVNNKKRPTVRHPTTPTNTAPNIGGFTPGGPTHNQRMQPITAESL RKERAY QKLLKKQQKEQDSLS KRHS KERAMLQKTHYSTVNKMEACHDREKLTREKS LEKAIK

KKGESNCSDLKIETEVHVQSLVNDHKARVKDWSDQSKEFADMVSRQQVEERSLKENHIQQR

CDLLKKLMLSTQEEQMQLLAGRNDREMKELKSNQAKDSMESTKSVSHDKSIKNKAERDRRVR

ELQSNNTKKFLEERKRLKMKQDEEKEEVQQEH

3. Amino acid sequence of sea urchin PLCp. Translated sequence of suPLCp with the characteristic PLC domains in bold and underlined. Each domain is labeled below the highlighted sequence. The * is above the site of Thrombin-mediated proteolysis. The sequence shaded in grey was used as the epitope for generating polyclonal antibodies. 46

Species Isoform # Identical % Identity # Homologous % Homology bovine PLCp4 629 55 804 70 C. elegans PLCp 601 52 837 73 human PLCpl 476 42 728 63 human PLCP2 404 35 666 58 human PLCp3 434 38 684 60 human PLCp4 555 48 683 60 hydra PLCpi 424 37 647 56 hydra PLCP2 437 38 705 61 lobster PLCp 584 51 808 70 mouse PLCpl 469 41 725 63 mouse PLCP3 436 38 697 61 mouse PLCp4 719 62 912 79 Drosophila NorpA 593 52 810 71 rat PLCpi 471 41 726 63 rat PLCP2 402 35 662 58 rat PLCp3 436 38 690 60 rat PLCP4 721 63 911 79 sponge PLCP 386 34 543 47 squid PLCP 430 38 699 61 Xenopus PLCp 451 39 714 62

fXgwre Comparison of suPLCf) to other known PLCP sequences. The listed sequences were individually aligned with suPLCp using ClustalW. The number of identical and homologous aligned amino acids was tabulated and a percentage was calculated based on the 1148 amino acids present in suPLCp. Each of the individual alignments used for this analysis may be found in the Appendix. 47

1 2 3 4 5 6 kDm

182.9

113.7

J. Generation of full-length smPLCP clone. The full-length coding sequence for suPLCp was cloned into the pcDNA3.1 expression vector (Invitrogen) and then added to a rabbit reticulocyte lysate reaction with ^S-methionine in order to visualize the translated product. Aliquots of the m wfro transcription/translation reactions were loaded onto 6% SDS-PAGE, proteins were then transferred to a nitrocellulose membrane, and the radio­ labeled bands were visualized after exposing the membrane to x-ray 61m. Lanes 1, 2, and 3 were loaded with 1, 2, and 5 p,l of the reticulocyte reaction, respectively, containing the full- length suPLCP coding sequence. Lanes 4, 5, and 6 were loaded with 1, 2, and 5 |il of the reticulocyte reaction, respectively, containing the expression vector alone as a negative control. The calculated molecular mass of the visible band is -134 kDa based on the standards located on the left of the image. 48

kDa 210 <* 131

89

42.2

B. 4 1 kDa 210 <* 131

89

42.2 + Blocking peptide - Blocking peptide

6. Anti-snPLCP Western blot analysis of jCyfec/WMw* protein lysates. Equal amounts of total protein were loaded onto 8% SDS-PAGE, transferred to nitrocellulose membranes, probed with anti-suPLCp antibodies followed by HRP-conjugated 2° antibody, and visualized via chemiluminescence on x-ray 61m. (A) Western blot of Z. /Hcfws soluble and particulate protein lysates from unfertilized eggs and at 1 minute post-fertilization, as well as whole sperm. (B) Western blots of 2. soluble and particulate protein lysates from unfertilized eggs and at 1 minute post-fertilization +/- blocking peptide in order to determine the specificity of the immunoreactive band(s). Lane 1: unfertilized soluble fraction, lane 2: unfertilized particulate taction, lane 3: 1 min. post-fertilization soluble taction, lane 4: 1 min. post-fertilization particulate taction, lane 5: whole sperm. The * denotes the anti-suPLCp immunoreactive protein. 49

kDa 210 *> 131

89

42.2

Blocking peptide + Blocking peptide fXgwre % Anti-suPLCP Western blot analysis of varwgaAfs protein lysates. Equal amounts of total protein were loaded onto 8% SDS-PAGE, transferred to nitrocellulose membranes, probed with anti-suPLCP antibodies followed by HRP-conjugated 2° antibody, and visualized via chemiluminescence on x-ray film. Western blots of L. variegatus soluble and particulate protein lysates from unfertilized eggs and at 1 minute post-fertilization +/- blocking peptide in order to determine the specificity of the immunoreactive band(s). Lane 1: unfertilized soluble fraction, lane 2: unfertilized particulate fraction, lane 3: 1 minute post- fertilization soluble fraction, lane 4: 1 minute post-fertilization particulate fraction. The * denotes the anti-suPLCP immunoreactive protein. 50

MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEGREGQVLE LSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMHMVASAPEI

AQVWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTR TFA8GKTEK1 VP KSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDIGRNETEGLSVEKLIEF

LNDKQRDPRLNEILFPMYDRERVLQI IDTYEKDP^II KKELLS SDGLCRYLMSDENAPVFLD

RLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYROVLLAGCRCVELDCWDGRSEDO EPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILSFENHCSKPQQLKLARYCEEVLGEF LLTEKLDDHPLDASVPLPS PNKLKRKILIKNKRLKPEVEQRQLELMRLRPELLEVNQEDEVE

ECES.JYS: UMT;:A;U;K:-KFI'MICV;:;-S : r:<;H:-. I I K:AKXI

i:f;g:'-;!^AvvAVYHYTo/x!

GYIKTQCIEFV§YNKRQMSR IYPQGRRYDS8NYMPQIFWNAGCQMVALNFQSPDIGMQLNQG

KIR-MÏCI -Û.M:K^VG:YVI -;V:Ï

my-j).jjHjt!-.iikïHï'VVA:\;i:,K]-vvKh1FJ'i-,vi-1HKvv:.„i n: /Wi.h iavyi-.ki i

PLDGIjQAGYRHISLRTEGNPPLSLPTVFCCIOLKTYVPDGLTgFVDALSDPQLYLSOAEKRA

KQLgCMG I BENE ISEVPTVNNKKRPTVRIIPTTPTNTAPNI GGFgPGGPTHNQRMQP ITAESL

NAY^X :: -K'-.V'V^.KW".• ;XI--.XVKAM: ,;;KV:Y ;: :VK;

KKGESNCSDLKI ETEVHVQSLV^HKAR\^DW8DQS KEFA^4V^RQOVEERSLI#fH IQQR

CDLLKKLMLSTQEE^QLLAGRNDREMKELKSNQAKDSMESTK^VSHDKSIKNXAERDRRVR . I • KMKQDEEKEEVQQEH

& Comparison of jCyfecAfMMs /wcf*» snPLCp with genome database. The 1. jpzcfug suPLCP sequence was used to do a BLAST search of the unannotated & genome database to find homologous sequence in this more distantly related species of sea urchin. The suPLCp sequence has regions highlighted in light gray where identical sequence was found in the database. Amino acids highlighted in dark gray indicate homologous amino acids were found in those positions within the database. 51

1 2 kDa 210 » f <* 131

89

42.2

P. Recombinant suPLCp expression and pnriGcation. Baculovirus-infected gyp cells were harvested and protein lysates were generated. Recombinant GST-suPLCp was puriGed torn the lysates using Glutathione-Sepharose 4B beads. Elution of the recombinant protein was then attempted using 20 mM Glutathione. Aliquots torn various steps of the purification protocol were saved to be run out on 8% SDS-PAGE, transferred to nitrocellulose membrane, probed with anti-suPLCp antibodies followed by HRP-conjugated 2° antibody, and visualized via chemiluminescence on x-ray film. Lane 1: total cell lysate from infected 6/P cells, lane 2: total cell lysate after incubation with Glutathione-Sepharose beads, lane 3: Glutathione-Sepharose beads after incubation with total cell lysate, lane 4: taction eluted from Glutathione-Sepharose beads with excess Glutathione (20 mM), lane 5: Glutathione-Sepharose beads after elution with excess Glutathione. The * indicates the anti- suPLCp immunoreactive recombinant GST-suPLCp with a molecular mass of-160 kDa. 52

4000

3500-

3000 -ï- ê& 2500 I 2000- Y 1500- à 1000 -f-

500

0 T T T control 420 nM 1 uM 2 uM 4 uM [GPy]

70. dose-response curve for suPLCP activation. Purited recombinant GST- suPLC|3 was incubated with a mixture of 3H-labeled PI(4,5)?2 in the absence (control) or presence of increasing concentrations of purited G^y and 2 uM tee calcium for 60 minutes at 37°C. The reactions were stopped by the addition of BSA and TCA to the reactions, which were then centrifuged to separate the soluble and insoluble tactions. The TCA- soluble taction was counted in a scintillation counter. The data represent the average of two independent experiments, each in triplicate ± SE. 53

4000

3500-

3000 i I& 2500- W

^ 2000-

^ 1500

1000 Ï

500

0 i » n y » | 1E-7 1E-6 1E-5 Free [Ca^] (M)

77. Calcium dependence of suPLCP activity. The PLC activity of purited recombinant GST-suPLCp was measured under calcium-EGTA buffering conditions designed to provide defined levels of free calcium. Purified recombinant GST-suPLCP was incubated with a mixture of ^H-labeled PI(4,5)Pz, 1 pM purified G^y, and the detned concentration of free calcium for 60 minutes at 37°C. The reactions were stopped by the addition of BSA and TCA to the reactions, which were then centrifuged to separate the soluble and insoluble tactions. The TCA-soluble taction was counted in a scintillation counter. The data represent the average of two independent experiments, each in triplicate ± SE. 54

A. kDa 210

13 i

<* 89 f

42.2 Jft

B. 1 2 3 4 5 kDa 210

131 m <* 89

42.2 m fXgwre 72. Thrombin-mediated proteolysis of GST-suPLCp. (A) After Thrombin proteolysis, the reactions were put through a Benzamidine column to remove the Thrombin. Eluted tactions were collected and run on 8% SDS-PAGE, transferred to nitrocellulose membranes, probed with anti-suPLCp antibodies followed by HRP-conjugated 2° antibody, and visualized via chemiluminescence on x-ray 61m. Lane 1 : Glutathione-Sepharose beads post-Thrombin proteolysis, lane 2: Elution #1, lane 3: Elution #2, lane 4: Elution #3. (B) Eluted tactions and Glutathione-Sepharose beads pre- and post-Thrombin proteolysis run on a 4-20% gradient gel followed by silver staining. Lane 1: Glutathione-Sepharose beads pre- Thrombin proteolysis, lane 2: Elution #1, lane 3: Elution #2, lane 4: Elution #3, lane 5: Glutathione-Sepharose beads post-Thrombin proteolysis. The * denotes the truncated suPLCp protein. 55

3000

2500 I 2000 & -f- 1"2 1500 1000-

500-

u "•—I— " T T control 420 nM 1 uM 2uM 4 uM [CM

73. G()y dose-response curve for activation of truncated suPLCP. PuriGed truncated suPLCP was incubated with a mixture of ^H-labeled PI(4,5)Pz in the absence (control) or presence of increasing concentrations of puriGed G|3y and 2 pM G-ee calcium for 60 minutes at 37°C. The reacGons were stopped by the addiGon of BSA and TCA to the reacGons, which were then centrifuged to separate the soluble and insoluble GacGons. The TCA-soluble G-acGon was counted in a scinGllaGon counter. The data represent the average of two independent experiments, each in duphcate ± SE. 56

3000

2500 ï

8 2000 & ï I1500- 1000

500

i i i i y y | ail»; a i i i i > y i | 1E-7 1E-6 ÎE-5 Free [Ca^] (M)

7^. Calcium dependence of truncated suPLCP activity. The PLC activity of puriGed truncated suPLCP was measured under calcium-EGTA buffering conditions designed to provide defined levels of tree calcium. Purified truncated suPLCP was incubated with a mixture of ^H-labeled PI(4,5)Pz, 1 puriûed G|3y, and the deGned concentration of free calcium for 60 minutes at 37°C. The reactions were stopped by the addition of BSA and TCA to the reactions, which were then centrifuged to separate the soluble and insoluble fractions. Hie TCA-soluble fraction was counted in a scintillation counter. The data represent the average of two independent experiments, each in duplicate ± SE. 57

suPLCP DVAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSSNYMPQIFW

Human PLCp2 EFSAQKNRSYVISSFTELKAYDLLSKASVQSypYNKRQMSRIYPKGTRMJJSSNYMPQMKW. .** • *********** . * * ******** . **

SuPLCp NAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSESPVDGVIA

Human PLCP2 NAC5CQMVALNFQTMBLPMQQKÎ4AVFEFNGQSGYLLKHEFMRRPDKQFNPFSVDRIDVWA ************. *. ** * **.** ***** .****** . *. * * * • * *•*

fXgwre 7J. Potential GPy-interaction site in snPLCp. Sequence alignment of human PLC(32 with suPLCp in the region shown in the human sequence to interact with G (By. The 62 amino acid binding region is highlighted in gray. 58

CHAPTER 5: DISCUSSION AND CONCLUSIONS

In this study I have for the first time shown by direct evidence that sea urchin eggs contain a PLC(3 isofbrm, which shall be referred to as suPLCp. To date the PLC isofbrms identified in sea urchins are the Ô (Coward ef a/., 2004) and the y (Shearer ef a/., 1999). A

Zyfec&mws pzcfws ovary cDNA library (Rakow and Shen, 1994) was screened and by using

5'- and 3'-RACE, a coding sequence ~3.4 kb in length, which translated into a 132 kDa protein, was generated.

Analysis of the translated sequence (Figure 3) indicated that the cloned protein contains the characteristic PH, EF-hand, X, Y, and C2 domains that are found in other PLCP enzymes (Rhee, 2001; Rebecchi and Pentyala, 2001), conûrming that this is a PLCp. Upon alignment with the amino acid sequences from different PLCp isofbrms, it was determined that suPLCp shares the highest degree of identity with PLCP4 (Figure 4). PLCP4 was first identified in bovine retina (Ferreira ef a/., 1993; Lee ef a/., 1993), in a mouse brain cDNA library (Lee ef a/., 1993) and in bovine cerebellum (Min ef o/., 1993). To date, expression of

PLCP4 remains limited to the retina and certain neuronal cells with the exception PLC-PH2 from hydra that has been suggested to be a homolog of vertebrate PLCp4/DroaopMo no/p/4 by phylogenetic analysis (Koyanagi ef a/., 1998). PLCp isofbrms have been identified in eggs from other organisms. In the mouse eggs, both PLCpi and PLCP3 have been identiGed

(Dupont ef a/., 1996; Wang ef g/., 1998). In Aenqpwf oocytes, a protein with the highest degree of similarity to PLCP3, referred to as PLC-XP, was identified (Ma ef a/., 1993). So, although there is evidence for PLCPs being present in the eggs of several organisms, this is 59 the first example of one similar to PLCfS4 being present in germ cells. The presence of suPLCp was verified in two other species of sea urchin, Zyfec&mws vanegaAw (Figure 7) and

(Figure 8). It can be concluded that suPLCp is not a protein that is unique to Z. /wcAw, but is most likely a conserved protein that is found in all sea urchins.

Western blot analysis using the anti-suPLCP antibodies indicated that there was a protein of the predicted molecular mass present in the egg lysates, but not in the sperm

(Figure 6A). The immunoreactive band is present in the particulate, or membrane fraction of the protein lysates but not the soluble, or cytosolic fraction. The location does not appear to change during fertilization. The only other PLC protein examined in sea urchin eggs is

PLCy, with two conflicting reports. In one case, immunocytochemistry performed with anti-

PLCy revealed the presence of this protein in cortical regions in the unfertilized egg (De

Nadai ef a/., 1998), while experiments with anti-PLCy immunoprecipitates of egg lysates that were fractionated using a sucrose gradient indicated that fertilization resulted in a translocation of the protein from the cytosol to the membrane (Rongish ef a/., 1999). The particulate localization is true specifically for PLC(34 (Lee ef o/., 1993). In general, PLCP isozymes are predominantly present in the particulate fraction when tissues and cultured cells are homogenized in a low ionic strength buffer (Lee ef o/., 1987; Jhon ef a/., 1993). Under more stringent conditions, PLCP can be found in the cytosolic fraction of bovine brain (Ryu ef a/., 1987). PLCy and PLC8 isozymes are detected mainly in the cytosolic fraction

(Rebecchi and Rosen, 1987; Ryu ef oA, 1987; Fukui ef a/., 1988; Homma ef o/., 1988). 60

Although the anti-suPLCP antibodies were utilized for Western blot analysis of egg protein lysates, sufficient quantities of the protein were not immunoprecipitated for enzymatic characterization. Recombinant suPLCp was expressed as a GST-fusion protein in

cells and purified for characterization of the enzymatic activity. PLC (34 enzymes, like all

PLCps, have their enzymatic activity regulated by heterotrimeric G proteins, although the other PLCp isofbrms can be activated by G protein a and Py subunits (Smrcka and

Stemweis, 1993; Boyer ef a/., 1994), PLCp4 enzymes are activated by G(%q, but they are not responsive to activation by GPy subunits (Jiang ef a/., 1994; Lee ef aA, 1994). Although suPLCp shares a high degree of identity with mammalian PLCP4s, based on the PLC activity assays, suPLCp can be activated by G(3y. There was consistently at least a 15-fbld increase in the enzymatic activity of suPLCp in response to GPy (Figure 13), with the concentration for half maximal activation (EC#) at about 500 nM. This response is similar to that of

PLCpi which had an EC# greater than 300 nM, while PLCP2 was about 30 nM, and PLCP3 was intermediate at about 90 nM (Smrcka and Stemweis, 1993). The greater EC# value for

PLCpi suggests it is probably not the primary means of regulating activity (Boyer ef a/.,

1994). Although the concentrations of G(3y required to activate PLCps in vitro are much higher than the effective concentrations of Goq subunits, the maximal extents of activation are similar (Rhee, 2001).

The activation of suPLCp by G protein Py-subunits was surprising due to its identity

with PLCP4, an isofbrm that has been shown not to be activated by GPy. One mechanism for

this mode of activation may be through the PH domain. Several intracellular signaling

proteins that contain the PH domain are also regulated by Gpy (Touhara ef oA, 1994; 61

Rebecchi and Scarlata, 1998). The PH domain has also been implicated as the site for GPy interactions in PLCps (Runnels and Scarlata, 1998; Wang ef a/., 1999; Wang ef a/., 2000).

There is also a second region that was first identified in PLCP2, spanning 62 amino acid

residues from Leu-580 to Val-641, that interacts with Gpy (Kuang ef a/., 1996). Subsequent

studies demonstrated that this was not only a site for binding but that binding was capable of

regulating the activity of the effector enzyme (Sankaran ef o/., 1998; Barr ef a/., 2000). Both

of these are potential sites for Gpy-mediated activation of suPLCp. There is a span of

sequence at the amino terminal end of suPLCp that has homology to PH domains found in

other PLCp proteins. There is also 77% homology for the 62 amino acid residue region

found in PLCP2 within the sea urchin sequence (Figure 15) which could act as a potential

candidate for GPy interaction.

As with other PLC enzymes, suPLCp does require Ca^ for its enzymatic activity.

Although other domains in PLC have the potential to bind Ca2+, a single Ca2+ ion bound in

the active site and coordinated by several amino acid side chains seems to be the only

essential metal, as shown in studies using PLCÔ (Grobler and Hurley, 1998). The hal&

maximal activity of suPLCp was detected at approximately 750 nM free calcium (Figure 14).

The calcium dependence exhibited by suPLCp is comparable to that of sea urchin PLCy

which has less than 10% of maximal activity detected at 200 nM free calcium and half-

maximal activity requiring 700-800 nM free calcium (Rongish ef a/., 1999). These calcium

concentrations are similar to what the enzyme would be exposed to in the egg. The [Ca^]t

within the unfertilized egg is -100 nM while the peak [Ca2^], level during the fertilization-

induced calcium transient is -2 pM (Shen, 1995). These levels would support minimal 62 activity of suPLCp within the unfertilized egg and would promote an increase toward maximal activity during the fertilization-induced transient.

To verify that the other prerequisite components of the signaling pathway are present in sea urchin eggs, Western blots of lysates using commercially available antibodies against

Goq were used, but these did not show any immunoreactive bands. Recently, Ga homologs, including Goq have been identified in sea urchin oocytes and egg (Voronina and Wessel,

2004). The data presented here indicates that a PLC(3 is present in the egg, localized to the plasma membrane where it would have access to its substrate, PIP;. 7» vzfro biochemical characterization of the enzymatic activity of suPLCp demonstrates that it can be regulated by

GPy dimers establishing a link to a heterotrimeric G protein pathway within the egg that would lead to an increase of IP3 generation. Based on the calcium-dependence of suPLCp activity, it is unlikely that this PLC participates in the small increase in IP3 mass immediately following fertilization as the [Ca^]j levels are low, but it may participate in the subsequent bulk increase in IP3 once the [Ca^]i have increased. 63

APPENDIX

Cwwpgrimwx of swfLC)) gegwence WA Âmwf* ALC)8 fgfwences. The amino acid sequence of suPLCp was aligned individually with each of twenty amino acid sequences of different PLCP isofbrms from eleven different species using ClustalW sequence alignment.

An * denotes conserved amino acid identity, while a : denotes amino acid homology in that position between the two sequences. 64

suPLCP MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEGREGQV bovine PLCP4

SUPLCP LELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMHMVAS

bovine PLCP4

SUPLCP APEXAQVWFDGL.LTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTRTFASGK

bovine PLCP4 PVRSITRTFASGK

** * e .********

suPLCP TEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDIGRNETEGL

bovine PLCP4 TEKVIFQALKELGLPSGKNDEIEPAAFTYEKFYELTQKICPRTDIEDLFKKINGDKTDYL

** * ; :*: ;*.******** ****** * .****** •********* ** * * J ;* ; *

suPLCP SVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSSDGLCRYLM

bovine PLCP4 TVDQLVSFLNEHQRDPRLNEILFPFYDAKRAMQIIEMYEPDEDLKKQGLISSDGFCRYLM :*:: * :.***; • ************.** .* .* * * . ** * .. *. *.****.*****

suPLCP SDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQVIiLAGCRCV

bovine PLCP4 SDENAPVFLDRLELYQEMDHPLAHYFISSSHNTYLTGRQFGGKSSVEMYRQVLLAGCRCV

****************. * . . * * . * * * * e ********************************

suPLCP ELDCWDGRSEDQEP11THGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILS FENHCS KPQQ

bovine PLCP4 ELDCWDGKGEDQEPIITHGKAMCTDILFKDVIQAIKETAFVTSEYPVILSFENHCSKYQQ

******* • p ********** ****** *.******.. ****** . . ************ **

suPLCP LKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNKRLKPEVEQRQLELM

bovine PLCP4 YKMSKYCEDLFGDLLLKQALESHPLEPGRPLPSPNDLKRKILIKKQTTETEVEKKQLEAL *...***...*..**. *.***. ****** ********.. » ***..***

suPLCP RLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIGSISGSSIAAEDE

bovine PLCP4 KSMMEAGESAAPVNMLEDDN--EEEIESAEQEEEAHPEYKYGNELSADDL-GHKEAIANS : * * .: * . :.

SUPLCP IKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNYAQPVKFQGFD

bovine PLCg4 VKKASDDLEHENSKKG-LVTVEDEQAWMASYKYVGATTNIHPYLSTMINYAQPVKFQGFH

I * : : * ; ** * * ; ; ** * * j*.*;*#*******# ** . . ***********

SuPLCP VAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRXYPQGRRYDSSNYMPQIFWN

bovine PLCg4 VAEERNIHYNMSSFNESVGLGYLKTHAIEFVNYNKRQMSRIYPKGGRVDSSNYMPQIFWN

******. * • *************. * * . i ****************. * * ************

suPLCP AGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSESPVDGVIAA

bovine PLCg4 SGCQMVSLNYQTPDLAMQLNQGKFEYNGSCGYLLKPDFMRRPDRTFDPFSETPVDGVIAA

:***** ; * * J *: ** : 4************ e**********************. ********

suPLCP TCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLNPVYNEEPFVFRK

bovine PLCP4 TCSVQVISGQFLSDKKIGTYVEVDMYGLPTDTIRKEFRTRMVMNNGLNPVYNEESFVFRK

****************. ***************** . * ^ *** * *********** < *****

SUPLCP WLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSLPTVFCCIQLK

bovine PLCP4 VILPDLAVLRIAVYDDNNKLIGQRILPLDGLQAGYRHISLRNEGNKPLSLPTIFCNIVLK *.************.. ^ .*********************** *** ******. * * * ** 65

suPLCP TYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIBENEISEVPTVNNKKRPTVRHPTT bovine PLCp4 TYVPDGFGDIVDALSDPKKFLSITEKRADQMRAMGIETSDIADVPSDTSKNDKKGKANTA

******. .. ******* . .* * .**** *. ** * * ;* • •**• *. e ; *•

suPLCP PTNTAPNIGG- FTP GGPTHNQR MQPITAESLRKERAYQKLLKKQQKEQDS bovine PLCg4 KANVTPQSSSELRPTTTAALGAGLEAKKGX ELIPQVRIEDLKQMKAYLKHLKKQQKELSS .*.*. »* * •. . . * *.. •** * ******* *

suPLCP LSKRHSKERAMLQKTHYSTVNKMEACHDREKLTREKSLEKAIKKKGESNCSDLKIETEVH bovine PLCp4 LKKKHAKEHSTMQKLHCTQVDKIVAQYDKEKLTHEKILEKAMKKKGGSNCLEMKKETEIK *__*.*.**. ; •** * • *.*• * .*.****•** ********* ** * • » * ***••

suPLCP VQSLVNDHKARVKDWSDQSKEFADMVSRQQVEERSLKENHIQQRCDLLKKLMLSTQEEQ bovine PLCP4 IQTLTSDHKSKVKEIVAQHTKEWSDMIHTHSAEEQEIRDLLLSQQCELLRKLLISAHEQQ .* . * ** * . •**• .* • • « •**• .**. ( . ^ ^ « .. . *,*.**.**.. * . .*.*

suPLCP MQLLAGRNDREMKELKSNQAKDSMESTKSVSHDKSIKNKAERDRRVRELQSNNTKKFLEE

bovine PLCP4 TQQLKLSHDRESKEMRAHQAKISMENSKAISQDKSIKNKAERERRVRELNSSNTKKFLEE * * .*** **....*** ** * .*.. * . ********** . ****** . * ********

suPLCP RKRLKMKQD---EEKEEVQQEH

bovine PLCP4 RKRLAMKQSKEMDQLKKVQLEHLEFVEKQNEQAKEMQQMVKLEAEMDRRPATW

**** ***p 66

suPLCp MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEGREGQV

C. elegans PLCP MAKEFQFNWKPTIIPELLHGSVFDRYDD-ESTCLELNAQVRIDENGFFLRWLIEGKDAW ** * • » *.*• • * .*..****•* .* .* .. * * ** * • • ** . . *

suPLCP LELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMHMVAS

C. elegans PLCP LDMGQIWEARTGGLPKDGRIMFELEQR GASETIAERTIWITHGQDLVWQSFFLVAE *::.** :.*.* *** : :: . .* :*** * *

SUPLCP APEIAQVWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTRTFASGK

C. elegans PLCp SVELAKTCRAGINDILKSSRIRHVCPTTQLIHTWLTMN--VNERRKIPIKLIIKTFSSGK : *: * :. : .:*** .* : ** . : :•*:***

suPLCP TEKIVFKSLDELGLPS GK MDEIDPADFPFEK

C. elegans PLCP PEKMVQKCLNDLGLGGDKYTPARVINRSMGKKFRNFYKCSRGRKRKEREELDVDILTFEK

* * . * * e * . . * * * * • • * • * • e * * *

suPLCP FYELYHKICPRTDIELLFKDIGRNETEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERV

C. elegans PLCP FQRLYNKICPRTEVQELFVKLS-GQKEYLTKERLXNFLNEEQRDPRLNEILFPFFDSQRI * ** • ****** • • - ** . • * *• *.**•***. .************ . . * .*.

SUPLCP LQIIDTYEKDPQMIKKELLSSDGLCRYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSH

C. elegans PLCP VALLKKHENDIKYQEDGKMSGDGFLRFLMSDENPPVFLDRIEMFMDMDQPLCHYYINSSH : :*.**: * ; ****** e ****** .* . . **.* * * 4 **.*****

suPLCP NTYLTGRQFGGKSSVEMYRQVLLAGCRCVELDCWDGRS EDQ-EP11THGIiAMCTDIQFRD

C. elegans PLCP NTYLTGRQYGGKSSSEIYRQVLLSGCRCIELDCWDGTGENKGEPIITHGKAMCTDVFFKD c ********. ***** * .****** .* * * * . ******* .*;; ******* *****. *.*

suPLCP VIQAIRDTAFVTSDFPVILSFENHCSKPQQLKLARYCEEVLGEFLLTEKLDDHPLDASVP

C. elegans PLCp VLVQIRDTAFARSDFPWLSFENHCSKSNQLKMAKYCMDIFGDMLLSKPFEDAPLDPGVS *. ****** *****. ********* •***.*.** ...*..**.. .. * ** * *

suPLCP LPSPNKLKRKILIKNKRLKPEVEQRQLELMRLRPELLEVN QED

C. elegans PLCP LPSPNRLRKKILIKNKRLKTDIERHQLDQFLREGKLDEEDELNETPEWGEDSVSPRSGG *****.*.. ********** ..*..**. . .* * . **

suPLCP EVEECESGYSDGAVSP P

C. elegans PLCp SGGTGAPEEVDDDTSDDDDDPSVQTSLNVMRTIPTVNTTSNNGSNRSARSSLDTPSPSGG

SuPLCP GNTDAHPKFKFPSKDS

C. elegans PLCP SLMVPDRATSTATSIKNAVLARSPNFSSLRQKLSFKRRQSPLAAGDQRAHPEVEQPVSSS

*• ** * . . * *

SUPLCP IGSISG SS IAAEDEIKLKHNMSVKMNAK

C. elegans PLCP SPATPSISGPPPCATSSGSTSSITITTTGCSTSSSGPSKHILGGEMPAKEMDEAHPELKQ **** ** - ; ** . ^ .* **

suPLCP KGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNYAQPVKFQGFD

C. elegans PLCP NFIAKNLKGFGFSKKQPVLTKEEEERIFAEYHYTGATTNIHPLLSSLVNYTHPVKFSGFD * ;* : : * : * :.*;*;*********. ** ** * *. .*******

suPLCP VAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSSNYMPQIFWN

C. elegans PLCP VAEANNLHFHMSSFSESTGLGYLKQSAPEFVNYNKRQSSRIYPKGARVDSSNFLPQIFWN *** ****.**** ** ****.* ^ ^ ********* *****. * * ****. . ****** 67

suPLCp AGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSESPVDGVIAA

C. elegans PLCP AGCQMVSLNFQTPDVYMQLNMGKFEYNGGSGYLLKPDFLRRPDRTFDPFSESPVDGVIAA ****** j* * * * . * * • **** ******* ******** • *********************

suPLCP TCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLNPVYNEEPFVFRK

C. elegans PLCP HCSVRVISGQFLSDRKIGTYVEVEMYGLPTDTIRKEHKTKVIPGNGLNPVYNEDPFVFRK

** * • ********* • * .****** ; ********** • ** • * • ^ # ********* • ******

suPLCP WLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSL-PTVFCCIQL

C. elegans PLCP WLPELAVLRFAVYDENGKQLGQRILPLDGLQAGYRHISLRSDTNQSFILSPVLFVQIVI **** . ***** . *** . * ^ ** . ******************** . . * • * * . * * •

suPLCP KTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIEENEISEVPTVNNKKRPTVRHPT

C. elegans PLCp KTYVPDELSGLVDALADPRAFLSEQKKRQEALAHMGVDDSDIPDVPNTRNMALRHVKQPP ****** *• .****.**. .**. .** • * **... .* .* * * *..*

suPLCP TPTNTAPNIG- -GFTPG GP THNQRMQPITAESLRKE

C. elegans PLCP RQNGSSADLLANNGQTGSARGDQTSSMASSTIRSPNEQPQPVAVDKFKVDPIEVDDLRRD

suPLCp RAYQKLLKKQQKEQDSLSKRHSKERAMLQKTHYSTVNKMEACHDR EKLTREKSLEK

C. elegans PLCP KAFAKLLKRFQKELDDLRKKHQKQRDSIQKQQQTNVDKLITNNRRSTKKEKGSR-RSLTA .*. ****. ** * * * *.* *•* .** . . *.*. . . * ** .* .**

suPLCP AIKK KGESN CSDLKIETEVHVQSLVNDHKA-

C. elegans PLCP SVSSGCGSASGTVTVSVCSPSGASCSGYSTGGPSTPVACNSDGTGSPATIGSPVPQDLVN ** * ..**.

suPLCp --RVKDWSDQSKEFADMVSRQQVEERSLKENHIQQRCDLLKKLMLSTQEEQMQLLAGRN

C. elegans PLCp NDRVRSLVNTQTGEWSAMVRRHDEEEFELKKVQLKEQFDLLRKLMSEAQKNQMLALKLRL **. • * * • *•• ** *.. ** **• ***.*** .*..** * *

suPLCP DREMKELKSNQAKDSMESTKSVSHDKSIKNKAERDRRVRELQSNNTKKFLEERKRLKMKQ

C. elegans PLCp EAEGKDLKQTQTKKSMEDAKVIQLDKGIKTKAERDRRVKELNEKNLKMFVEERKRLAMKA • * *•** ****** .* . ** ** ********. * * . .* * *.****** **

suPLCP DEEKEEVQQEH

C. elegans PLCp QKHEEQLTKRHLDQLEQLDKDFHKALDAEVGNYKEEQLAAQPTSW 68

suPLCp MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEG

Human PLCpi MAGAQPGVHALQLKPVCVSDSLKKGTKFVKWDD-DS-TIVTPIILRTDPQGFFFYWTDQN ** * . ** . * • * • • •* ** ... * ***.*.

suPLCp REGQVLELSQXNDVRKG---IAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNI

Human PLCpi KETELLDLSLVKDARCGRHAKAPKDP KLRELLDVGNIGRLEQRMITWYGPDLVNI

•* .. * . * * .. * * * ** * * *. . * e *.:*:* **: * *; * * *

suPLCp NYMHMVASAPEIAQWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNL

Human PLCpi SHLNLVAFQEEVAKEWTNEVFSLATNLLAQNMSRDAFLEKAYTKLKLQVTPEGRIPLKNI ....** *. * . * ...... *. *. * . *. * . *« •* * *.****. • *•

suPLCP TRTFASGKTEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDI

Human PLCpl YRLFSADR--KRVETALEACSLPSSRNDSIPQEDFTPEVYRVFLNNLCPRPEIDNIFSEF

* * * .: * : .***.: *.* **. * : :

suPLCP GRNETEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSS

Human PLCpi GAKSKPYLTVDQMMDFINLKQRDPRLNEILYPPLKQEQVQVLIEKYEPNNSLARKGQISV * • *.*.....*.* ***********.* .*.* .*. ** . . .* .*

suPLCP DGLCRYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQV

Human PLCpi DGFMRYLSGEENGWSPEKLDLNEDMSQPLSHYFINSSHNTYLTAGQLAGNSSVEMYRQV

** ; ** * * j •*•* .** ***************** *. ^ *. *********

suPLCP LLAGCRCVELDCWDGRSEDQEPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILSFE

Human PLCpi LLSGCRCVELDCWKGRTAEEEPVITHGFTMTTEXSFKEVXEAIAECAFKTSPFPILLSFE **. ********** **. ..**.****• .* *;*.*: ;**;** ; ** ** **..****

suPLCP NHCSKP-QQLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNKRLKPE

Human PLCpi NHVDSPKQQAKMAEYCRLIFGDALLMEPLEKYPLESGVPLPSPMDLMYKILVKNKKRSHK ** .. * ** *• * ** ..*. ** * *. .**.. ****** * ***.***.

suPLCp VEQRQLELMRLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIGSXS

Human PLCpi SSEG SGKKK- - -LSEQASNTYSDSSS MFEPSSPGAGEAD . : : :

suPLCP GSSIAAEDEIKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNYA

Human PLCpl TESDDDDDDD DCKK SSMDEGTAGS EAMAT EEMSNLVNYI .* :* : :.** * :* . ****

suPLCP QPVKFQGFDVAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSS

Human PLCpi QPVKFESFEISKKRNKSFEMSSFVETKGLEQLTKSPVEFVEYNKMQLSRIYPKGTRVDSS ****** *. . . . .** *.**** *. ** . •***.*** *.*****.* * ** *

suPLCP NYMPQIFWNAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSE

Human PLCpi NYMPQLFWNAGCQMVALNFQTMDLAMQINMGMYEYNGKSGYRLKPEFMRRPDKHFDPFTE *****. ************** . *. ** . * * .**** ** ***.******. ****.*

suPLCP SPVDGVIAATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLNPVY

Human PLCPl GIVDGIVANTLSVKIISGQFLSDKKVGTYVEVDMFGLPVDTRRKAFKTKTSQGNAVNPVW ***..* * **.. ******************* . *** ** *. .*.* * .* * * .

suPLCP NEEPFVFRKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSLP

Human PLCPl EEEPIVFKKWLPTLACLRIAVYEEGGKFIGHRILPVQAIRPGYHYICLRNERNQPLTLP .***.**.***** ** ***********. ** . **** . . ^ .. **. . * ** * * **.** 69

suPLCP TVFCCIQLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIEE

Human PLCPl AVFVYIEVKDYVPDTYADVIEALSNPIRYVNLMEQRAKQLAALTLEDEBSVKKEADPGET • ** *. . * ** * * .. • »***.* *. *.***** . • *«

suPLCP NE ISBVPTVNNKKRPTVRHPTTPT NTAPNIGGFTPGGPTHN QRMQP

Human PLCpi PSEAPSEARTTPAENGVNHTTTLTPKPPSQALHSQPAPGSVKAPAKTEDLIQSVLTEVEA

.* . * : * . * . . * : : : * * * . :

suPLCP ITAESLRKERAYQKLLKKQQKEQDSLSKRHSKERAMLQKTHYSTVNKMEACHDREKLTRE

Human PLCpi QTIEELKQQKS FVKLQKKHYKEMKDLVKRHHKKTTDLIKEHTTKYNEIQNDYLRRRAALE * * * * ** . ** * ** * * . . * * * » * • . • . * ^ . • *

suPLCP KSLEKAIKKKGE-SNCSDLKIETEVHVQSLVNDHKARVKDWSDQSKEFADMVSRQQVEE

Human PLCPl KSAKKDSKKKSEPSSPDHGSSTIEQDLAALDAEMTQKLIDLKDKQQQQLLNLRQEQYYSE ** •* ** * * * * • * • *• * ... * *

suPLCP RSLKENHIQQRCDLLKKLMLSTQEEQMQLLAGRNDREMKELKSNQ-AKDSMESTKSVSHD

Human PLCpi KYQKREHIKLLXQKLTDVAEECQNNQLKKLKE X CEKEKKELKKKMDKKRQEKITEAKS KD • * • ** • * . *••*•• * • •* ** * * . * *• . *. *

suPLCP KS 1KNKAERDRRVRELQSNNTKKFLEERKRLKMKQDEEKE EVQ

Human PLCpi KSQMEEEKTEMIRSYIQEWQYIKRLEEAQSKRQEKLVEKHKEIRQQILDEKPKLQVELE ** * ; « ; .* . * ** . . . **

suPLCP QEH

Human PLCpi QEYQDKFKRLPLEILEFVQEAMKGKISEDSNHGSAPLSLSSDPGKVNHKTPSSEELGGDI

** :

suPLCp

Human PLCpi PGKEFDTPL 70

SUPLCP MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEGREGQV

Human PLC02 MSLLNPVLLPPKVKAYLSQGERFIKWDD--ETTVASPVILRVDPKGYYLYWTYQSKEMEF » ** * -

suPLCP LELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMHMVAS

Human PLC(32 LDITSIRDTRFGKFAKMPKSQKLRDVFNMDFPDNSFLLKTLTWSGPDMVDLTFHNFVSY . * . * . * * * • * •

SUPLCp APEIAQWJFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTRTFASGK

Human PLC|32 KENVGKAWAEDVLALVKHPLTANASRSTFLDKILVKLKMQLNSEGKIPVKNFFQMFPADR •* • * .*•***»*«

SUPLCP TEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDIGRNETEGL

Human PLCp2 --KRVEAALSACHLPKGKNDAINPEDFPEPVYKSFLMSLCPRPEIDEIFTSYHAKAKPYM * * •* ** ** * *•* *** • • •***.*• •*

suPLCP SVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSSDGLCRYLM

Human PLCP2 TKEHLTKFINQKQRDSRLNSLLFPPARPDQVQGLIDKYEPSGINAQRGQLSPEGMVWFLC *. * •*.*•**** *** .*** .s * .** ** * * • * • • *

suPLCP SDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQVLLAGCRCV

Human PLCpz GPENSVLAQDKLLLHHDMTQPLNHYFINSSHNTYLTAGQFSGLSSAEMYRQVLLSGCRCV ** • ; *. * *..** ***_ ************* ** * ** ********.*****

suPLCp ELDCWDGRSEDQEPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILSFENHCSKP-Q

Human PLCP2 ELDCWKGKPPDEEP11THGFTMTTDIFFKEAIEAIAESAFKTS PYPIILSFENHVDSPRQ ******. *.******* . . * ** * *; . *. * * ..** ** .*.******* ,.* *

suPLCP QLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPS PNKLKRKILIKNKRLKPEVEQRQLEL

Human PLCp2 QAKMAEYCRTIFGDMLLTEPLEKFPLKPGVPLPSPEDLRGKILIKNK KNQFSG * *. * ** ..*..**** *. ** ******. *. ******* . *.

suPLCP MRLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIGSISGSSIAAED

Human PLCP2 PTS SSKD TGGEAEGSS - PP - -S - - APAVWAGEE GTELEEEE *..*. ** . * . *.. *.

suPLCP EIKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNYAQPVKFQGF

Human PLCP2 VEEEEEEESGNLDEEEIKKMQS-DEG TAGLEVTAYEEMSSLVNYIQPTKFVSF : * :: : :: : : **. :* .. : .********* *

suPLCP DVAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSSNYMPQIFW

Human PLCp2 EFSAQKNRSYVISSFTELKAYDLLSKASVQFVDYNKRQMSRIYPKGTRMDSSNYMPQMFW . * • . * * * * .;**.***********.* * ***********

suPLCP NAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSESPVDGVIA

Human PLCP2 NAGCQMVALNFQTMDLPMQQNMAVFEFNGQSGYLLKHEFMRRPDKQFNPFSVDRIDVWA ************. *. ** * ** . * * ***** .******. *.*** .* *.*

suPLCP ATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVP-NNGLNPVYNEEPFVF

Human PLCp2 TTLSITVISGQFLSERSVRTYVEVELFGLPGDPKRR-YRTKLSPSTNSINPVWKEEPFVF

:* *; ********. . * ***•**...*** * ** ;* * ; * *e .***. .******

suPLCP RKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSLPTVFCCIQ

Human PLCP2 EKILMPELASLRVAVMEEGNKFLGHRIIPINALNSGYHHLCLHSESNMPLTMPALFIFLE

*::;*;** ** : ** *** *..*.**.*.. *. .**.*. *. . * *. ** ..*..* 71

suPLCP LKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMG--IEENEISEVPTVNNKKRPTVR

Human PLCgZ MKDYIPGAWADLTVALANPIKFFSAHDTKSVKLKEAMGGLPEKPFPLASPVASQVN-GAL

:* * :* . . : :: . * * : :* ::* :* : :. ...* .: .

suPLCP HPTTPTNTAPNIGGFTPGGPTHNQRMQPITAESLRKERAYQKLLKKQQKEQDSLSKRHSK

Human PLCP2 APTSNGSPAARAGAREEA-MKEAAEPRTASLEELRELKGWKLQRRHEKELRELERRGAR

**: * :. : *. ** : :. ** .* . : * ::

suPLCP ER- -AMLQKTHYSTVNKMEACHD REK- - LTREKSLEKAIKK KG

Human PLCP2 RWEELLQRGAAQLAELGPPGVGGVGACKLGPGKGSRKKRSLPREESAGAAPGEGPEGVDG « * * * • * • ** • * • * * ** . * * • *

suPLCp ESNCSDLKIETEVHVQS--LVNDHKARVKDWSDQSKEFADMVS-RQQVEERSLKENHIQ

Human PLCP2 RVRELKDRLELELLRQGEEQYECVLKRKEQHVAEQISKMMELAREKQAAELKALKETSEN

. . . :: * *: *. : * *: : * :* .* ::***. :

suPLCP QRCDLLKKLMLSTQEEQMQLLAGRND REMKELKSNQAKDSMESTKSVSHDKSIKN

Human PLCP2 DTKEMKKKLETKRLERIQGMTKVTTDKMAQERLKREINNSHIQEWQVIKQMTENLERHQ •• *** * . * *. *...... *•. ..

suPLCP KAERDR RVRELQSNNTKKFLEERK-RLKMKQDEEKEEVQQEH

Human PLCP2 EKLEEKQAACLEQIREMEKQFQKEALAEYEARMKGLEAEVKESVRACLRTCFPSEAKDKP

: *: * * : *: * ; * **.*•_

suPLCP

Human PLCP2 ERACECPPELCEQDPLIAKADAQESRL 72

suPLCP --MAKQYVFQWQVCVP EPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEG

Human PLCg3 MAGAQPGVHALQLEPPTWETLRRGSKFIKWDEETSS--RNLVTLRVDPNGFFLYWTGPN *: *. *: * * :: : : *. .: :: ** ***:*:.. .

suPLCP REGQVLELSQINDVRKG---IAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNI

Human PLCp3 MEVDTLDISSIRDTRTGRYARLPKDP-KIREVLGFGGPDAR--LEEKLMTWSGPDPVNT *• *.. * * * * * *** . * * .*...*.*****

suPLCP NYMHMVASAPEIAQWFDGLLTITHKTVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNL

Human PLCP3 VFLNFMAVQDDTAKVWSEELFKLAMNILAQNASRNTFLRKAYTKLKLQVNQDGRIPVKNI * . * - ** • * • • . *• * « * *«* • *• •*** •*****.*.

SuPLCp TRTFASGKTEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDI

Human PLCP3 LKMFSADK--KRVETALESCGLKFNRSESIRPDEFSLEIFERFLNKLCLRPDIDKILLEI : ** :*:* *.**; ;*

suPLCP GRNETEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSS

Human PLCp3 GAKGKPYLTLEQLMDFINQKQRDPRLNEVLYPPLRPSQARLLIEKYEPNQQFLERDQMSM * • *• .*.*.*********.*.* . •*. ** . * *

suPLCP DGLCRYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQV

Human PLCgS EGFSRYLGGEENGILPLEALDLSTDMTQPLSAYFINSSHNTYLTAGQLAGTSSVEMYRQA ;* ; ** * ; *; * ;* ** **** ************ *• *^ ********

suPLCP LLAGCRCVELDCWDGRSEDQEPIXTHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILSFE

Human PLCP3 LLWGCRCVELDVWKGRPPEEEPFITHGFTMTTEVPLRDVLEAIAETAFKTSPYPVILSFE ** ******** * ** .•**.****. .* *.. .***..** .*** ** .*******

suPLCP NHCS-KPQQLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPS PNKLKRKILIKNK-RLKP

Human PLCP3 NHVDSAKQQAKMAEYCRSIFGDALLIEPLDKYPLAPGVPLPSPQDLMGRILVKNKKRHRP

** , ** *;*e**f e .. * . ** * ** •* * ******. * .**.*** * .*

suPLCP EVEQ RQLELMRLRPELLEX7NQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKD

Human PLCp3 SAGGPDSAGRKRPLEQSNSALSESSAATEPSSPQLGSPSSDSCPGLSNGEEVGLEKPSLE * ... .* ... . :: :

SuPLCp SIGSISGSSIAAEDEIKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLS

Human PLCP3 PQKSLGDEGLNRGPYVLGPADREDEEEDE--EEEEQTDPKKPTTDEGTASSEVNATEEMS *• • » » ...* ** •< ..* •*

suPLCP RLVNYAQPVKFQGFDVAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQG

Human PLCP3 TLVNYIEPVKFKSFEAARKRNKCFEMSSFVETKAMEQLTKSPMEFVEYNKQQLSRIYPKG ** * * ;****• .*; * •** ****** *. . ;***;***;•;*****•*

SuPLCp RRYDSSNYMPQIFWNAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRT

Human PLCP3 TRVDSSNYMPQLFWNVGCQLVALNFQTLDVAMQLNAGVFEYNGRSGYLLKPEFMRRPDKS * ********. *** ***. ****** . *. ** * * * ***** ^ ******. ****** . .

SUPLCP FDPFSESPVDGVIAATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNN

Human PLCP3 FDPFTEVIVDGIVANALRVKVISGQFLSDRKVGIYVEVDMFGLPVDT-RRKYRTRTSQGN ****** ***..* . *. ********* . * * * ******. * * * ^ ** **..**** *

suPLCP GLNPVYNEEPFVFRKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGN

Human PLCP3 SFNPVWDEEPFDFPKWLPTLASLRIAAFEEGGKFVGHRILPVSAIRSGYHYVCLRNEAN

e .***. .**** * ***** ** ** * * .***** . .*.****. _ ^ .. . * * . . . ** * * 73

suPLCf) FPLS LPTVFCCIQLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIE-ENEISEV

Human PLCp3 QPLCLPALLIYTEASDYIPDDHQDYAEALINPIKHVSLMDQRARQLAALIGESEAQAGQE

**.**::: : . *:**. :* :: * ::**:** .: * * : .:

suPLCP PTVNNKKRPTVRHPTT-PTNTAPNIGGFTPGGPTHNQRMQPITAESLRKERAYQKLLKKQ

Human PLCp3 TCQDTQSQQLGSQPSSNPTPSPLDASPRRPPGPTTSPASTSLSSPGQRDDLIASILSEVA

* *** . . * :

suPLCP QKEQDSLSKRHSKERAMLQKTHYSTVNKMEACHDREKLTREKSLEKAIKK-KGESNC

Human PLCP3 PTPLDEL--RGHKALVKLRSRQERDLRELRKKHQRKAVTLTRRLLDGLAQAQAEGRCRLR ** * * *. • ... *.*..* .* 0,.* *

suPLCP -SDLKIETEVHVQSLVNDHKARVKDWSDQSKEFADMVSRQQVEERSLKENHIQQRCDLL

Human PLCP3 PGALGGAADVEDTKEGEDEAKRYQEFQNRQVQSLLELREAQVDAEAQRRLEHLRQALQRL

.* . :* . : *

suPLCP KKLMLSTQEEQMQLLAGRNDREMKELKSNQAKDSMESTKSVSHDKSIKNKAER---DRRV

Human PLCP3 REWLDANTTQFKRLKEMNEREKKELQKILDRKRHNSISEAKMRDKHKKEAELTEINRRH

*::* *:*****;- .* *..** .**

suPLCp RELQSNNTKKFLEERK RLKMKQDEEKEEVQQEH

Human PLCP3 ITESVNSIRRLEEAQKQRHDRLVAGQQQVLQQLAEEEPKLLAQLAQECQEQRARLPQEIR ** *: :

suPLCP

Human PLCP3 RSLLGEMPEGLGDGPLVACASNGHAPGSSGHLSGADSESQEENTQL 74

suPLCP MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEGREGQV Human PLCP4

suPLCP LELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMHMVAS

Human PLCP4

suPLCP APEIAQVWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTRTFASGK

Human PLCP4 MNNNWNVCFFLFCP SI TRTFASGK

* e * *********

suPLCp TEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDIGRNETEGL

Human PLCP4 TEKVIFQALKELGLPSGKNDEIEPTAFSYEKFYELTQKICPRTDIEDLFKKINGDKTDYL

***. .*. .* ******** ***.*. *i .****** .********* ** * *, *

suPLCP SVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSSDGLCRYLM

Human PLCP4 TVDQLVSFLNEHQRDPRLNEILFPFYDAKRAMQIIEMYEPDEDLKKKGLISSDGFCRYLM

:*.:***: ** * :: ** *;****;*****

suPLCP SDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQVLLAGCRCV

Human PLCP4 SDENAPVFLDRLELYQEMDHPLAHYFISSSHNTYLTGRQFGGKSSVEMYRQVLLAGCRCV

****************; * ; . ** ; * * * * e ********************************

suPLCp ELDCWDGRSEDQEPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILSFENHCSKPQQ

Human PLCP4 ELDCWDGKGEDQEPIITHGKAMCTDILFKDVIQAIKETAFVTSEYPVILSFENHCSKYQQ *******. ^ ********** ****** *•******• .******• .************ **

suPLCP LKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNKRLKPEVEQRQLELM

Human PLCP4 YKMSKYCEDLFGDLLLKQALESHPLEPGRALPSPNDLKRKILIKNKRLKPEVEKKQLEAL *. . ;***; .:*; :**.: ***** *****************; •*** .

suPLCP RLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIGSISGSSIAAEDE

Human PLCP4 RSMMEAGESASPANILEDDN--EEEIESADQEEEAHPEFKFGNELSADDLG * * * . *.

suPLCP IKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNYAQPVKFQGFD

Human PLCp4 HKEAVANSVKKG-LVTVEDEQAWMASYKYVGATTNIHPYLSTMINYAQPVKFQGFH *. •* ** * *.. ** * * .* *.* ******* ** .. ***********

suPLCP VAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSSNYMPQIFWN

Human PLCp4 VAEERNIHYNMSSFNESVGLGYLKTHAIEFVNYNKRQMSRIYPKGGRVDSSNYMPQIFWN

******.*.*************.**. ****************;* * ************

suPLCP AGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSESPVDGVIAA

Human PLCP4 AGCQMVSLNYQTPDLAMQLNQGKFEYNGSCGYLLKPDFMRRPDRTFDPFSETPVDGVIAA **»***;**;*;**; ************ **********************.********

suPLCP TCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLNPVYNEEPFVFRK

Human PLCp4 TCSVQVISGQFLSDKKIGTYVEVDMYGLPTDTIRKEFRTRMVMNNGLNPVYNEESFVFRK ****************.*****************.* ^ *** * *********** ^ *****

suPLCP WLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSLPTVFCCIQLK

Human PLCP4 VILPDLAVLRIAVYDDNNKLIGQRILPLDGLQAGYRHISLRNEGNKPLSLPTIFCNIVLK *. ************ . . ^ *********************** ^ *** ********* * ** 75

suPLCp TYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIEENEISEVPTVNNKKRPTVRHPTT

Human PLC(34 TYVPDGFGDIVDALSDPKKFLSITEKRADQMRAMGIETSDIADVPSDTSKNDKKGKANTA ******. • ********* .* * .**** *. ** * * .*..**. *• . *.

suPLCP PTNTAPN IGGFTPGGPTHN- -QRMQPITAESLRKERAYQKLLKKQQKEQDS

Human PLCP4 KANVTPQSSSELRPTTTAALASGVEAKKGIELXPQVRIEDLKQMKAYLKHLKKQQKELNS

suPLCP LSKRHSKERAMLQKTHYSTVNKMEACHDREKLTREKSLEKAIKKKGESNCSDLKIETEVH

Human PLCP4 LKKKHAKEHSTMQKLHCTQVDKIVAQYDKEKSTHEKILEKAMKKKGEVIVSK *. * I * 2 ** ! I ;* * * • *• * • * .*.** *.** ********** *

suPLCp VQSLVNDHKARVKDWSDQSKEFADMVSRQQVEERSLKENHIQQRCDLLKKLMLSTQEEQ

Human PLCP4

suPLCP MQLLAGRNDREMKELKSNQAKDSMESTKSVSHDKSIKNKAERDRRVRELQSNNTKKFLEE

Human PLCP4

suPLCP RKRLKMKQDEEKEEVQQEH

Human PLCP4 76

suPLCP ---MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSE--G

Hydra PLCPl MAGAGPISETLPVISVPPMLVNGGRFLKWDDQD--LTCAIYILEVDKLGHVLSWKPKDFS * * * * * * . . . * *

suPLCP REGQVLELSQINDVRKG IAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDF

Hydra PLCpl KDTEVLELVLVRDVRTGQYAKIPKAQDTRARDAIKMYSAVELESNLMQERTITIVYGSNF . **** . *** * .***** * • *

suPLCP VNINYMHMVASAPEIAQWFDGLLTITHMVKANNVCPMQSLKKHWMKICFQVNPNGRIPV

Hydra PLCPl VDLTFINFVASSVSEAQEWTEALFKCTNNLLEFHSSPLKCLERFYTRICVQKNQNGEIPV * ** * - ** * • *• *.*. .** * * ** ** *

suPLCP RNLTRTFASGKT-EKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELL

Hydra PLCpi KILVKAFVNHKEDIKRAYGTLQSLGIAHGKKDMIVLKDFSFETFFTFYNRLISRHDFDNV • * ••* * * • •*• **. ** * * ** ** *• .*... **..

suPLCP FKDIGRNETEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKE

Hydra PLCPl FTELGAKNKPYLTTDQLVEFLNNRQRDPRLNEILFPYYNINAADAIVNTYEPNKSFADKG * • •* • • *• • •*.****• .************ *.

suPLCP LLSSDGLCRYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEM

Hydra PLCpl LISIDGLMKFLMSNDANVLLPGKLNIHQDLSFPIAHYFINSSHNTYLQGHQLTGKSSVEI *.* ** * ..***. .*...**. *..************ *.*. ******.

suPLCP YRQVLLAGCRCVELDCWDGRSEDQEPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVI

Hydra PLCpi YRQVLLAGCRCIELDCWDGKTEEQEPVITHGMTLCTEVLFKDVIEAIKESAFKTSEYPVI ***********. ******* . .*.***.****. ..**. . *.***•**• ..** **..***

suPLCP LSFENHCSKPQQLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNKRL

Hydra PLCpi LS FENHCSAKQQMKMANYCLQIFGDLLLNKELDEYPLLPGTLLPSPKVLKRKILIKNK-- ******** **.*.*** :: :**.: :**: ;* * ... ** * * ; **********

suPLCp KPEVEQRQLELMRLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIG

Hydra PLCpl - -KREIKDTKNANISTQSVDLN SSGRFEP • *...... * ** *

suPLCP SISGSSIAAEDEIKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLV

Hydra PLCPl EVLNIEEIP EDAEDLSKSEEEEAPEVEAQ--Q ELSELV

auPLCP NYAQPVKFQGFDVAEERNLHFNMSS FNESVGLGYIKTQCIE FVNYNKRQMSRIYPQGRRY

Hydra PLCpi NYFQPVHFRGFEKAEKRKCYYEMSSFGENTATNLLKEQPVEFVNYNKFQNSRIYPKGSRV ** ***•*.**. **.*. •••**** * .* * .******* * *****.* *

SUPLCP DSSNYMPQIFWNAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDP

Hydra PLCpi SSDNYIPQLFWNAGCQMVALNFQTLDVPMQLNLGKFEINGRCGYLLKPEHMRRHDLTFDP

„ *. * * ; * * ; ************** . *. ** * * ** * * ** *******. ** * * ** * *

suPLCP FSESPVDGVIAATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLN

Hydra PLCpi FVDSTVDGIIAGTVQVKVISGQCLLERRSGTYVEVEMYGLPADTVRRKYRTKWSNNGLN * .****.*** *.***** * ... ******. ***** ;**; ** ; . * * . ******

suPLCP PVYNEEPFVFRKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPL

Hydra PLCpi PVYDEDPFKF-KVILPHLAELRIAVYEENGKMIGHRILPVDGLLPGFRHIKLRNESYQPL

***;* ; * * * ** :# * ********_**.* *.* * * *.* * * *.****** ** 77

suPLCP SLPTVFCCIQLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIEENE--ISEVPT

Hydra PLCPl CLPTLFVDIVTKDYVPESFSDFADALSNPMKYLSEKEKRTQQLEAFEFEESDSTALQVPT ** * • * * * ** * • • • •* ****** ** * • ** * • •* * • .* * » •* * *

suPLCP W-NKKRPTVRHPTTPTNTAPNIGGFTPGG-PTHNQRMQPITAESLRKERAYQKLLKKQQ

Hydra PLCpl TNKNILKVTVQEPQNLVKHGSKISLQADSYQGQKDKYVAEPMKQTKRKRSQSQSLGRLES

.* * : **:.* . .: : . :: : : :: **. *.* : : .

suPLCP KEQDSLSKRHSKERAMLQKTHYSTVNKMEACHDREKLTREKSLEKAIKKKGESNCSDLKX

Hydra PLCPl PKQP--IRRTFSTPKLLGEDTSSDYLKISGLSIN--TLKEHKLYKKWKLKFDSDFLNLMQ » * • * • * * * • • * . * * * * . * . . *

suPLCP ETEVHVQSLVNDHKARVKDWSDQSKEFADMVSRQQVEERSLKENHIQQRCDLLKKLMLS

Hydra PLCPl KQAKGRIQCRKEIEAKTKKLTLNLE K KNQLYKR-1KTNFLKQQ- KLMEIIMKY • e . e *. * • » * .* .* * *• . .*

suPLCP TQEEQMQLLAGRNDREMKELKSNQAKDSMESTKSVSHDKSIKNKAERDRRVRELQSNNTK

Hydra PLCPl TITVQLK

SUPLCP KFLEERKRLKMKQDEEKEEVQQEH

Hydra PLCPl 78

SUPLCP MAKQYVFQWQV--CVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEGREG

Hydra PLC#2 MQNKFFNAIEINDKVKEVFQKGVYVKKLEE-DK-SSISTCLLKIDAFGFFLSYQEKDKDA * ... .. ** ... * .* • * * •**.*.* •* ** • *• •

suPLCP QVLELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMHMV

Hydra PLCgZ FCIDICDISYVKDGATPKALNKVFEQYSDEEKNN-VDFLSRFTYICFGTNIVEVSFITFD

*: .* :** :. : : * : . ** * :

suPLCP ASAPEIAQVWFDGLLTITHNVKANNVCP--MQSLKKHWMKICFQVNPNGRIPVRNLTRTF

Hydra PLCP2 FMTKELKQIFFSCISTNLTNHKHAFRNPSVKQMLYKQYVLSKSMVNASGKISMKNIFKCL • * • * . . * . * * * * * * * . . , ** * « * . . * ; • •

suPLCp ASGKTEKIVFKSLD-ELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDIGRN

Hydra PLCP2 NAVRFEQTTIEGX QTALSLPVG---EIDPEMFS FEQFYKLCCLVCPRPDIDELFISLFG-

• j *. p ** * ** * * .*** **. ** •

suPLCP ETEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSSDGL

Hydra PLC#2 --TRLTAEKLMKFLNEDQRDPRLNEQMHPHCTISNAVDLIMDYESNEACRTKELLTIQGF *• ***. •***• ******** . * ...* ** . ****- .*.

suPLCP CRYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQVLLA

Hydra PLCpz IQLLLSKESAVLNPECLLLRHDMNQPINHYFINSSHNTYLLGRQVYGKSSVEIYRQVLIS • *:*.*.* ; : * * .**.**. ************ ** * ******.*****..

suPLCP GCRCVELDCWDGRSEDQEP11THGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILS FENHC

Hydra PLCP2 GCRCIELDCWDGPG VP11THGKAMCTNVLFSDWQAIADTAFLTS PYPWLS FENHC ** * * • ******* ****** ****.. * **.*** ****.** .**. *******

suPLCp SKPQQLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNKRLKPEVEQR

Hydra PLCP2 SLPQQKILAEMCIKlFGPLLLDKPLESNPLEPGIELPSPEQLKYKILIKNKKLASNDEMQ * ** * ** * ...* .** . *• •**. . ****..** *******. * . * .

suPLCp Q LELMRLRPEL LEVNQEDEVEECESG

Hydra PLCP2 FSNGKLNSSANISYDIPVEMPETNTSSYSSCDVPDLESESEMEHDNCFNEDDLSRMVTED

: : **

suPLCP YSDGAVS - - PPG NTDAH

Hydra PLCg2 NFETKLNEKMDQFAKQVYSESSSNNQPVMRRRSTQEFNLKKYKLSKIAENCWDDSEDEI . *

suPLCp PKFKFPSKDSIGSIS GSSIAAEDEIKLKHNMSVKMNAKKGELLSQEDEAAWA

Hydra PLCp2 ILTKFGSNPCIASALNEEVNSNTTTSVLECEDNSSHFPSKWRSHSVAPLNQSETEAVDIK ** * • * * • • * * * * * * **

suPLCP QYRY TGATTNIHACLSRLVNYAQPVKFQGFDV

Hydra PLCgZ RRAISDISDIETSKKVSSMSTVMSNLPDNSIITSVSGKVDPLLSSLINYVQPQSFKGFEY

suPLCP AEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSSNYMPQIFWNA

Hydra PLC^S AEEQKKHYIMSSFSELEGLKQCTENLTDFVLYNKRQFSRIYPKATRVESTNYVPQVFWNA

** * : : *; ****.* ** , ; :** *****;*****: * .*.**.**.** * *

suPLCp GCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSESPVDGVIAAT

Hydra PLCp2 GCQFVALNLQTPDLSVQLNQQKFECNFNCGYLLKPVKYRETDSNFDPLTENPIDGIIPLT ***.*•**.*.**. _ .**** ** * * ******* * * ** * . .**.**.* * 79

suPLCP CSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLNPVYNEEPFVFRKV

Hydra PLC(Î2 VEIEVISGQYLSEKKCMTYVQIDMFGLSADTTRKK-RTNLSESNLMNTVYPKTVFKFDKI

suPLCP VLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSLPTVFCCIQLKT

Hydra PLCPZ VFPEMVLVRFTVYDESDKLLGQRVLPLEGIKSGYRHIAMRTETGHFLPLTTLFVKFTFKI *• *• t .. * . . * * . * **•***•***•*• • • *****. . * * * * * *«* • • *

suPLCP YVPDGLTQFVDALSDPQ LYLSQAEKRAKQLLCMGIEE NEIS

Hydra PLCPZ YIHEKYEDAVEELFNPNKYKLLAEHRASRIAKKLEDMLLSDEDIIVQNPKLIQICGDQAN

* : : : * : * : * : * : : : .

suPLCP EVPTVNNKKR PTVRHPTTPTNTAPNIG GFTPGGPTHNQRMQPITAES

Hydra PLCp2 STPTETKRKSSVSSMGSSLNHPSLITSNTIDNSSIKMKSFSLTFDESNMGNRMLELCEDE

suPLCP LRKER AYQKLLKKQQKEQDSLSKRHS KERAMLQKTHYSTVNKMEACHDREK

Hydra PLCp2 FLKERKIDDFKLDKMFLRIAKKNGKEVDILKQNHRKERKLTESFYDEEISKLQMYYDKTL » ** * . « ; ** • ** * * . * ** * . . . ^ . * . . . * .

suPLCP LTREKSLEKAIKKK--GESNCSDLKIETEVHVQSLVN

Hydra PLCpz ESELKSFEKSLQKINTKEVQASYQKIVEDVSIKFDKSSRKIIDSQMMELKEKYLEKTKIW • **•**...* * • * ** .* ..

suPLCp --DHKARVKDWSDQSKEFADMVSRQQVEERSLKENHIQQRCDLLKKLMLSTQEEQMQLL

Hydra PLCPZ KTEKVGKISVIVEEQKKALKIMIDEHIEKEARIKYSHSIQQIDLIGKLLKTAQDNDIKKL

:: :* :* .* *: **: **: *

suPLCP AGRNDREMKELKSNQAKDSMESTKSVS HDKSIKNKAERDR RVRELQSNNT

Hydra PLCPs AEINEKELSDLTKAQARDSIQSIKDLEKEFENSKMDRINKEKIKRERDKLRXEEFGMQRR * *••*..* **•**••* * . *. *.*.*•* *. *•

suPLCP KKFLEERKR LKMK QDEEKEEVQQEH

Hydra PLCps KLVQNQQKRQEKLIEKHKNENKQFEDHRKEIIQLGYEPIIRHIRQAQTLISLHQQKL * ; ; ; ** * * . * ** .* . 80

suPLCp MAKQYVFQWQVCVPEPLKNGAMFDRYE-DGDSGSVEQSCLFKVDEYGFFIYYKSEGREGQ

Lobster PLCp MTKYFDFVWRKEVDSRLQEGAVIDRWTEDKESYDYEPNCTFKVDEYGFFVFWKSDGREGN

*.* . * *. * e *.; * * ; ; * * ; * ;* . * .* *********; : . * * . * * * * ;

suPLCP VLELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMHMVA

Lobster PLCP VAELSQVSDIRRGTLPRDSKLLDKL---LNKFG-QDVEDKMLAICSGLDYVSINYINIVC * ****. *; *. * *; * * *; ; ** ;; * * ; ;- ****•*;*.***; ; ; * .

suPLCp SAPEIAQVWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTRTFASG

Lobster PLCp KTKDEADAWLEGLRNIFHNIRANNICPMMCLKKHWMRLGMMVDSGGKIPVKHIVRTFASG *. *..** * **. .***.*** ******. . . *• *.***. ; • ******

SuPLCP KTEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDIGRNETEG

Lobster PLCP KTEKMVYSCLEEMGLPSGKGDSIEKDAFTFEKFYKLYHTICPRTDIDELYTSVTKG--EH

****;*. *.*.****** * *. *,***** j ** * e*******. *

suPLCP LSVERLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSSDGLCRYL

Lobster PLCP VSLAQLVNFMNEKQRDPSLNEILYPLYDEKRCMQIIKAHEPNEENIENKRFSKDGLLAYL

.* . .*..*•*.***** *******.** # • * .* * * • .* j • *. . . •* ** * **

suPLCP MSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQVLLAGCRC

Lobster PLCP MSDENDPVFLDRLDIYQDMDQPLSHYYINSSHNTYLSGRQFGGKSTAEMYRQTLLAGCRC

***** ******* .. * * * * ; ****** . ********* . ******** j *****a*******

suPLCP VELDCWDGRSEDQEPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILSFENHCSKPQ

Lobster PLCP VELDCWDGKGEDEEPIITHGKAMCTDILFKEAIIAIRDCAFVTSDYPVILSFENHCCKKQ ******** ; **. ******* ****** * ; ; * ** * * ****** - ********** ,* *

SUPLCP QLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNKRLKPEVEQRQLEL

Lobster PLCP QYKLAKYCDEYFGDLLSKEALPDRPLVPGHHLPSPNQLKRKILIKNKRLKPEVEKQELEL * ***;**.* .*..* * * *.** ***** ****************** • • .***

suPLCP MRLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIGSISGSSIAAED

Lobster PLCp F-WKGQLYLVDDDD AREDASQANP QPKEPKSNSCTTITTPVSQPSLED : : :* * :: : * :**

suPLCP EIKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNYAQPVKFQGF

Lobster PLCP D-K PLTAEER AFLYYQYTGATTHVHPVLSSYVNYCQPVNFQGF * *..* *.******.. * ** *** ********

suPLCP DVAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSSNYMPQIFW

Lobster PLCP DVAKEKNVHHNMSSFSETMGLGYLKTQAIEFVNYNKRQMSKlYPKGARVDSSNYMPHVFW ***.*.*.* ***** *. .****.*** ************.*** .* * ******* • - **

suPLCP NAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSESPVDGVIA

Lobster PLCP NAGCQMVSLNFQTSDLPMQLNQGKFEYNGNCGYLLKPDFMRRTDKTFDPFAESPVDGVIA ******* - ** * * . * j ************ ************ ^ *. ***** . *********

suPLCp ATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTR- -TVPNNGLNPVYNEEPFV

Lobster PLCP AQVSVSVIAGQFLSDKKVGTYVEVDMYGLPTDTIRKEFRTRYDSLEMALIPSIMKNLPPF * ** ** • **************************. * ** * .. . . • . *

suPLCP FRKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSLPTVFCCI

Lobster PLCp LSCVCLTWLCCELVCFMKIEKYWAKEFCLWMVCMQAGYRHISLRTEGNFPMSLPMLFCNI

• * * * . . . * . .**************** . * * * .* * * 81

suPLCP QLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIBENEIS EVPTVNNKKRPTVRH

Lobster PLCP ELKIYVPDGLGDLMDALSDPRAFLSAQEKREEQMKAMGIEASDIDTKALKGGGGKAGGAK .** ****** .. . ****** . .** ** * •*• ** * *

suPLCp PTTPTNTAPN-IGGFTPGGP--THNQRMQPITAESLRKERAYQKLLKKQQKEQDSLSKRH

Lobster PLCP PAAGAKAGACGKPGACAGGKKKKKFVEYEPINLETLRSQKNFLKATKKQQKELESMRKKH • * * * • ** * ****** •*• *« *

suPLCP SKERAMLQKTHYSTVNKMEACHDREKLTREKSLEKAIKKKGESNCSDLKIETEVHVQSLV

Lobster PLCP MKERLSIQK A QCSAIEKLAK G — K—KDVMDD- - - ** * • **

suPLCP NDHKARVKDWSDQSKEFADMVSRQQVEERSLKENHIQQRCDLLKKLMLSTQEEQMQLLA

Lobster PLCP PKIKSLVSEQMKQWSEMMEKHRKEEWEMLKEHLKSQEDILKKLMEEEQAGQIKKME • * .* * . * *. *. ***** * *.

suPLCP GRNDREMKELKSNQAKDSMESTKSVSHDKSIKNKAERDRRVRELQSNNTKKFLEERKRLK

Lobster PLCP AKHEQDMKDMKAQQAKAAMETAKDVNQDKTLKTKADRDRRLKEKNQNWTKRFIEERKISA **..*..*** .**..* * .**••* **.****.. * • ****.*•****

suPLCP MKQDEEKEEVQQEH

Lobster PLCp MKQGKQKEKLKKVHDQQMTELTKDIQMAIQFYENTEAEYKMWNKMEFFC ***. i ;**; ; ; i * 82

suPLCP MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEG

Mouse PLCPl MAGAQPGVHALQLKPVCVSDSLKKGTKLVKWDD-DS-TIVTPTILRTDPQGFFFYWTDQN .. ***•**.*. . ...* ***.*. .

suPLCP REGQVLELSQINDVRKG IAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNI

Mouse PLCPl KETELLDLSLVKDARCGKHAEAPKDP KLRELLDVGNIGHLEQRMITWYGPDLANI .* ••*;** .. * * * **** *. . * e *.«*.* ** . * * • ^ **

suPLCp NYMHMVASAPEIAQVWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNL

Mouse PLCPl SHLNLVAFQEEVAKEWTNEVFSLATNLLAQNMSRDAFLEKAYTKLKLQVTPEGRIPLKNI ....** *.*. * • ..... *. * .* . *•* • *• •** *.****.•*•

suPLCP TRTFASGKTEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDI

Mouse PLCpi YRLFSADR--KRVETALEACSLPSSRNDSIPQEDFTPDVYRVFLNNLCPRPEIDNIFSEF

* .***.: *.* **. : : :

suPLCP GRNETEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSS

Mouse PLCpi GAKSKPYLTVDQMMDFINLKQRDPRLNEILYPPLKQEQVQVLIEKYEPNSSLAKKGQMSV * • *•* *»* ************* .*.* •*• ** . . ** .*

suPLCP DGLCRYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQV

Mouse PLCPl DGFMRYLSGEENGWSPEKLDLNEDMSQPLSHYFINSSHNTYLTAGQLAGNSSVEMYRQV **. ** * .* * ^ * ..*.* .** ^ *****************^ *. *. *********

suPLCP LLAGCRCVELDCWDGRSEDQEPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILSFE

Mouse PLCpl LLSGCRCVELDCWKGRTAEEEPVITHGFTMTTEISFKEVIEAIAECAFKTS PFPILLSFE ************* **• ..**.****. .* *.* *..**.** . ** ** **..****

suPLCP NHCSKP-QQLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNKRLKPE

Mouse PLCPl NHVDSPKQQAKMAEYCRLIFGDALLMEPLEKYPLESGVPLPSPMDLMYKILVKNKKKSHK

** ** *.***, ..*. ** * *. .**.. ****** * *•*•***. ^ j

suPLCp VEQRQLELMRLRPELLEVNQEDEVEECESGYSDGAVS PPGNTDAHPKFKFPS KDSIGSIS

Mouse PLCPl SSEG SGKKK- - -LSEQASNTYSDSSSVFE PSSPGAGEAD . : : : * : .. . ***. :

suPLCP GSSIAAEDEIKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNYA

Mouse PLCpi TESDDDDDDD DCKK SSMDEGTAGSEAMAT EEMSNLVNYI .* :* ; ;.* * *. **. : . :: * .*****

suPLCP QPVKFQGFDVAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSS

Mouse PLCPl QPVKFESFEISKKRNKSFEMSSFVETKGLEQLTKSPVEFVEYNKMQLSRIYPKGTRVDSS

*****..*; I I I .** *;**** *. ** ; ** * ; ** * *.*****.* * ** *

suPLCP NYMPQIFWNAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSE

Mouse PLCPl NYMPQLFWNAGCQMMALNFQTVDLAMQINMGMYEYNGKSGYRLKPEFMRRPDKHFDPFTE

*****. ******** . ***** . *. **.* * .* * * * e* * ** * . ****** . ****.*

suPLCP SPVDGVIAATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLNPVY

Mouse PLCpi GIVDGIVANTLSVKIISGQFLSDKKVGTYVEVDMFGLPVDTRRKAFTTKTSQGNAVNPVW ***..* * **. .A******************.*** ** *. *.* * .***.

suPLCP NEEPFVFRKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSLP

Mouse PLCpi EEEPIVFKKVVLPSLACLRIAAYEEGGKFIGHRILPVQAIRPGYHYICLRNERNQPLTLP .***.**.***** ** ** * * ******. ** . **** . . .. **..* ** * * ** . * * 83

suPLCP TVFCCIQLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIEENE---ISEVPTVN

Mouse PLC(3l AVFVYIEDKDYVPDTYADVIEALSNPIRYVNLMEQRAKQLAALTLEDEEEVKKEADPGET • ** *. * **** .***•* *• ******* • .*..*

suPLCP NKKRPTVRHPTTPTNTAPNIGGFTPGGP THNQ RMQP

Mouse PLCpl SSEAPSETRTTPAENGVNHTASLAPKPPSQAPHSQPAPGSVKAPAKTEDLIQSVLTEVEA

suPLCP ITAESLRKERAYQKLLKKQQKEQDSLS KRHSKERAMLQKTHYSTVNKMEACHDREKLTRE

Mouse PLCpi QTIEELKQQKS FVKLHKKHYKEMKDLVKRHHKKTTELIKEHTTKYNEIQIDYLRRRAALE ** **• ** * ** * *. * * *

suPLCP KSLEKAIKKKGE-SNCSDLKIETEVHVQSLVNDHKARVKDWSDQSKEFADMVSRQQVEE

Mouse PLCPl KSAKKDSKKKSEPSSPDHGSSAIEQDLAALDAEMTQKLIDLKDKQQQQLLNLRQEQYYSE ** .* *** * *

SuPLCg RSLKENHIQQRCDLLKKLMLSTQEEQMQLLAGRNDREMKELKSNQ-AKDSMESTKSVSHD

Mouse PLCPl KYQKREHIKLLIQKLTDVAEECQNNQLKKLKEICEKEKKELKKKMDKKRQEKITEATSKD

• * ** * *

suPLCP KS IKNKAERDRRVRELQSNNTKKFLEERKRLKMKQDEEKEE

Mouse PLCPl KSQMEEEKTEMIRSYIQEWQYIKRLEEAQSKRQEKLVEKHNEIRQQILDEKPKGEGPSS .*. * **• .*..*..

suPLCP VQQEH

Mouse PLCPl VLSEGCHEDPSVPPNFTPPNPQALKW * . * 84

suPLCP --MAKQYVFQWQVC---VPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEG Mouse PLCp3 MAGARPGVHALQLEPSTWKTLRRGSKFIKWDEEASS--RNLVTLRVDPNGFFLYWTGPN * . * * . * • * • * • * • •* * ***.*.

suPLCP REGQVLELSQINDVRKG IAPKDVGYLAKVGSIPDPFGRGNIEDRTXTICSGLDFVNI

Mouse PLCP3 MEVDTLDISSIRDTRTGRYARLPKDP-KIREVLGFGGPDTR--LEEKLMTWAGPDPVNT *. *..***** *** • •* * * . * . . - * • • * * * *

suPLCP NYMHMVASAPE IAQVWFDGLLTITHNVKANNVCPMQS LKKHWMKICFQVNPNGRIPVRNL

Mouse PLCp3 TFLNFMAVQDDTVKWSEELFKLAMNILAQNASRNTFLRKAYTKLKLQVNQDGRIPVKNI .** . *. .. *. *. * *. * • * • .* * * .*****•*;

suPLCP TRTFASGKTEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDI

Mouse PLCP3 LKMFSADK--KRVETALESCGLNFNRSESIRPDEFPLEIFERFLNKLCLRPDIDKILLEI * . • * * * • * • * * * * • * * • * * » • * • * * * * •

suPLCP GRNETEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSS

Mouse PLCP3 GAKGKPYLTLEQLMDFINQKQRDPRLNEVLYPPLRSSQARLLIEKYETNKQFLERDQMSM *•.*.*•.*.*•*********•*•*

suPLCP DGLCRYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQV

Mouse PLCp3 EGFSRYLGGEENGILPLEALDLSMDMTQPLSAYFINSSHNTYLTAGQLAGPSSVEMYRQA .* . ** * .** . *. *. * ** ** * * ************ *« * ********

suPLCP LLAGCRCVELDCWDGRSEDQEP XITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILSFE

Mouse PLCP3 LLWGCRCVELDVWKGRPPEEEPFITHGFTMTTEVPLRDVLEAIAEAAFKTSPYPVILSFE ** ******** * ** ..**•****• • * *.. .***••** ..** ** .*******

suPLCP NHCS-KPQQLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNK-RLKP

Mouse PLCP3 NHVDSAKQQAKMAEYCRSIFGDALLIDPLDKYPLSAGIPLPSPQDLMGRILVKNKKRHRP ** ** *.* ** • « *. ** . ** • ** * .*****. * .**.*** * .*

suPLCP EVEQRQLELMRLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIGSI

Mouse PLCP3 STGVP-DSSVRKRPLEQSNSALSESSAATEPSSPQLGSP--SSDSCPGLSNGEEVGLEKT * * * . * . *

SuPLCP SG--SSIAAEDEXKLKHNMSVKMNAKKGELLSQEDE AAWAQYRYTGATTNIHAC

Mouse PLCP3 SLEPQKSLGEESLSREPNVPMPDRDREDEEEDEEEEETTDPKKPTTDEGTASSEVNATEE

suPLCP LSRLVNYAQPVKFQGFDVAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYP

Mouse PLCp3 MSTLVNYVEPVKFKSFEASRKRNKCFEMSSFVETKAMEQLTKSPMEFVEYNKQQLSRIYP . * ** * * .****. *. . .** *.**** * . •***•***•*.*****

suPLCP QGRRYDSSNYMPQIFWNAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPD

Mouse PLCP3 KGTRVDSSNYMPQLFWNVGCQLVALNFQTLDLPMQLNAGVFEYNGRSGYLLKPEFMRRPD

;* * ********; *** . *** ; ****** • *; **** * ***** i****** .******

suPLCP RTFDPFSESPVDGVIAATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVP

Mouse PLCp3 KS FDPFTEVIVDGIVANALRVKVISGQFLSDKKVGIYVEVDMFGLPVDT- RRKYRTRTSQ ..****.* ***..* . *. ************* ******. *** ** **..****

suPLCP NNGLNPVYNEEPFVFRKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTE

Mouse PLCP3 GNSFNPVWDEEPFDFPKWLPTLASLRIAAFEEGGKFVGHRILPVSAIRSGYHYVCLRNE * »***..**** * ***** ** ** * * .***** . .*.****. ...**... ** * 85

suPLCP GNFPLSLPTVFCCIQLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIEENEISE

Mouse PLCP3 ANQPLCLPALLIYTEASDYIPDDHQDYAEALINPIKHVSLMDQRAKQLAALIGESEAQAS

.* **.**::: : . *:**. :* :: * ::***** .: *.; :.

suPLCp VPTVNNK--KRPTVRHPTTPTNTAPNIGGFTPGGPTHNQRMQPITAESLRKERAYQKLLK

Mouse PLCP3 TETYQETPCQQPGSQLPSNPTPNPLDASPRWPPGPTTSSTSSSLSSPGQRDD-LIASILS

. * ::* : *:.** . * *** ..

suPLCP KQQKEQDSLSKRHSKERAMLQKTHYSTVNKMEACHDREKLTREKSLEKAIKKK-GESNC-

Mouse PLCP3 EVTPTPLEELRSH-KAMVKLRSRQDRDLRELHKKHQRKAVALTRRLLDGLAQARAEGKCR

: . : * *

suPLCP -SDLKIETEVHVQSLVNDHKARVKDWSD-QSKEFADMVSRQQVEERSLKENHIQQRCDL

Mouse PLCP3 PSPSALGKATNSEDVKEEEEAKQYREFQNRQVQSLLELREAQADVETKRKLEHLRQAHQR

* *,*:*::* :

suPLCP LKKLMLSTQEEQMQLLAGRNDREMKELKSNQ---AKDSMESTKSVSHDKSIKNKAERDRR

Mouse PLCp3 LKEWLDTHTTQFKRLKELNEREKKELQKILDRKRNNSISEAKTREKHKKEVELTEINRR

*:: * *:** ***:. : :* ;**

suPLCP VRELQSNNTKKFLEERK RLKMKQD EEKE EVQQEH -

Mouse PLCp3 HITESVNSIRRLEEAQKQRHERLVAGQQQVLQQLEEEEPKLLAQLTQECQEQRERLPQEI *. :: ; * :* ** *; **.* ..**

suPLCP

Mouse PLCP3 RRCLLGETAEGLGDGPLVACASNGHAPGSGGHLSSADSESQEENTQL 86

suPLCp MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEGREGQV

Mouse PLCP4 MAKPYEFNWQKEVPSFLQEGAVFDRYEE-ESFVFEPNCLFKVDEFGFFLTWKSEGKEGQV * * * * * . * * * * *••**.*****. .* * ******* . * * * . **********

SUPLCP LELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMHMVAS

Mouse PLCp4 LECSLINSIRQAAIPKDPKIXiAALEAVGKSE--NDLEGRILCVCSGTDLVNIGFTYMVAE ** * ** •*. *** ** .* * . •*** *.*** . .***

suPLCp APEIAQVWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTRTFASGK

Mouse PLCP4 NPEVTKQWVEGLRSIIHNFRANNVSPMTCLKKHWMKLAFLTNTTGKIPVRSITRTFASGK **... * .** . * * * • * * * * * * ******* . * * ****** . ********

suPLCp TEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDIGRNETEGL

Mouse PLCP4 TEKVIFQALKELGLPSGKNDEIEPAAFTYEKFYELTQKICPRTDIEDLFKKINGDKTDYL * * * * ;*; ; * , ******** ****** * . ****** * ********* * * * * ^ ; • * : *

suPLCP SVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSSDGLCRYLM

Mouse PLCP4 TVDQLVSFLNEHQRDPRLNEILFPFYDAKRAMQIIEMYEPDEELKKKGLISSDGFCRYLM .*. * * . * * * * * ************ • ** .* .***. * * * . . * * *.****.*****

suPLCP SDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQVLLAGCRCV

Mouse PLCP4 SDENAPVFLDRLELYQEMDHPLAHYFXSSSHNTYLTGRQFGGKSSVEMYRQVLLAGCRCV **************** . *. . * * • ** * * t ********************************

suPLCP ELDCWDGRSEDQEPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILSFENHCSKPQQ

Mouse PLCP4 ELDCWDGKGEDQEP11THGKAMCTDILFKDVIQAIKETAFVTSEYPVILS FENHCSKYQQ ******** ********** ****** *.******. . ****** . . ************ * *

suPLCP LKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKHKRLKPEVEQRQLELM

Mouse PLCp4 YKMSKYCEDLFGDLLLKQALESHPLEPGRPLPSPNDLKRKILIKNKRLKPEVEKKQLEAL *;..***...*..** . * . * * * . ****** ***************** . .***

suPLCP RLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIGSISGSSIAAEDE

Mouse PLCp4 KSMMEAGESAAPASILEDDN--EEEIESADQEEEAHPEYKFGNELSADDYS * « •***..** . * *

suPLCP IKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNYAQPVKFQGFD

Mouse PLCP4 HKEAVANSVKKG-LVTVEDEQAWMASYKYVGATTNIHPYLSTMINYAQPVKFQGFH * ; J * ^ ** * *. . ** * * .* * .* ******* ** .. ***********

suPLCP VAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSSNYMPQIFWN Mouse PLCp4 VAEERNIHYNMSS FNESVGLGYLKTHAIEFVNYNKRQMSRIYPKGGRVDSSNYMPQIFWN ******.*.*************.**. ****************.* * ************

suPLCP AGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSESPVDGVIAA

Mouse PLCP4 AGCQMVSLNYQTPDLAMQLNQGKFEYNGSCGYLLKPDFMRRPDRTFDPFSETPVDGVIAA ******. * * • * . * * . e ************ ^ ********************** . ******** suPLCp TCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLNPVYNEEPFVFRK Mouse PLCP4 TCSVQVISGQFLSDKKIGTYVEVDMYGLPTDTIRKEFRTRMVMNNGLNPVYNEESFVFRK **************** . ***************** . * * * * * *********** *****

suPLCP WLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSLPTVFCCIQLK Mouse PLCp4 VILPDLAVLRIAVYDDNNKLIGQRILPLDGLQAGYRHISLRNEGNKPLSLPTIFCNIVLK * ; ************ . . i _ *********************** ^ *** ********* * * * 87

BuPLCg TYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIEENEISEVPTVN--NKKRP---TV

Mouse PLCj34 TYVPDGFGDIVDALSDPKKFLSITEKRADQMRAMGIETSDIADVPSDTSKNDKKGKANPA ******. .. ******* . .** .* * * * *. **** .*..**. * *.

suPLCP RHPTTPTNTA PNIGGFTPGG PTHNQRMQPITAESLRKERAYQKLLKKQQKEQDS

Mouse PLCP4 KANVTPQSSSELRPTTTAALGSGQEAKKGIELIPQVRIEDLKQMKAYLKHLKKQQKELNS

I .* * , ; ; *, . .* , ; ; I * *; ; *********** .*

suPLCP LSKRHSKERAMLQKTHYSTVNKMEACHDREKLTREKSLEKAIKKKGESNCSDLKIETEVH

Mouse PLCP4 LKKKHAKEHSTMQKLHCTQVDKIVAQYDKEKSTHEKILEKAMKKKGGSNCLEIKKETEIK * *•*•**•. » ** * • *.*. * .*•** *.** ********* ** * • .* ***..

suPLCP VQSLVNDHKARVKDWSDQSKEFADMVSRQQVEERSLKENHIQQRCDLLKKLMLSTQEEQ

Mouse PLCP4 IQTLTTDHKSKVKEIVAQHTKEWSEMINTHSAEEQEIRDLHLSQQCELLRKLLINAHEQQ • *•* ***..**•.*....**...*. 0 ** . ... *. *.*.**.**.. ..*.*

SUPLCP MQLLAGRNDREMKELKSNQAKDSMESTKSVSHDKSIKNKAERDRRVRELQSNNTKKFLEE

Mouse PLCP4 TQQLKLSHDRESKEMRAHQAKISMENSKAISQDKSIKNKAERERRVRELNSSNTKKFLEE * * .*** **....*** ** * •*. .************ . ****** .* ********

suPLCP RKRLKMKQD EEKEEVQQEH

Mouse PLCp4 RKRLAMKQSKEMDQLKKVQLEHLEFLEKQNEQAKEMQQMVKLEAEMDRRPATW

** * * ** * # ; ; J ;* * ** 88

suPLCP MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDS GSVEQSCLFKVDEYGFFIYYKSEGR

Drosophila NorpA MTKKYEFDWIIPVPPELTTGCVFDRWFENEKETKENDFERDALFKVDEYGFFLYWKSEGR

*• *; * *; * ; ** * * • ** * . . ; *• # ********** .* . *****

SUPLCP EGQVLELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMH

Drosophila NorpA DGDVIELCQVSDIRAGGTPKDPKILDKVTKK-NGTNIPELDKRSLTICSNTDYINITYHH •*»*.** *. *•* * .*** ** * • ... *..**** *..** **

SUPLCP MVASAPEIAQWFDGLLTXTHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTRTF

Drosophila NorpA VICPDAATAKSWQKNLRLITHNNRATNVCPRVNLMKHWMRLSYCVEKSGKIPVKTLAKTF

• •. . *• * * ** * * .* ** * * * ****.. - *. *.***. *..**

suPLCP ASGKTEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDIGRNE

Drosophila NorpA ASGKTEKLVYTCIKDAGLPDDKNATMTKEQFTFDKFYALYHKVCPRNDIEELFTSITKGK *******.*. ^ ^ . _ . ** * e * . .**.*** ** * * . * * * ^ ** * ** * . .

suPLCP TEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERVXjQXXDTYEKDPQMIKKELLSSDGLC

Drosophila NorpA QDFISLEQFIQFMNDKQRDPRMNEILYPLYEEKRCTEXINDYELDEEKKKNVQMSLDGFK ! •*•*••*•*•********.****•*.*• .* .**. ** * . *. .* **.

suPLCP RYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQVLLAG

Drosophila NorpA RYLMSDENAPVFLDRLDFYMEMDQPLAHYYINSSHNTYLSGRQIGGKSSVEMYRQTLLAG ****************..* .*.***.**.*********.***.*********** ****

suPLCP CRCVELDCWDGRSEDQEPIITHGLAMCTDXQFRDVIQAIRDTAFVTSDFPVILSFENHCS

Drosophila NorpA CRCVELDGWNGKGEDEEPIVTHGHAYCTEILFKDCIQAIADCAFVSSEYPVILSFENHCN *********. * . **.***.*** * **.* *.* ** * * * ***.*. ***********

suPLCP KPQQLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNKRLKPEVEQRQ

Drosophila NorpA RAQQYKLAKYCDDFFGDLLLKEPLPDRPLDPGLPLPPPCKLKRKILIKNKRMKPEVEKVE ;.* * ***.**.. ;*; ;**,* * *.*** .*** * ************. ***** . .

suPLCP LELMRLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIGSISGSSIA

Drosophila NorpA LELWLKG ELKTDDDPEE DASAGKPPEAAAAP APAPEAAAAAEG A *** *: : :* : ** * :: * * *: :: .: .* *

SUPLCP AEDEIKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNYAQPVKF

Drosophila NorpA AE GGGG AEAEAAAAN YSGSTTNVHPWLSSMVNYAQPIKF

** . * ****>s *.*.***.* ** .******.**

suPLCp QGFDVAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSSNYMPQ

Drosophila NorpA QGFDKAIEKNIAHNMSSFAESAGMNYLKQSSIDFVNYNKRQMSRIYPKGTRADSSNYMPQ

**** * *.*. _ ***** **„*; .*: * e*;**************.* * ********

suPLCP IFWNAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSESPVDG

Drosophila NorpA VFWNAGCQMVSLNFQSSDLPMQLNQGKFEYNGGCGYLLKPDFMRRADKDFDPFADAPVDG .*********.***** *. ************ ************ *. ****...****

suPLCP VIAATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLNPVYNEEPF

Drosophila NorpA VIAAQCSVKVIAGQFLSDKKVGTYVEVDMFGLPSDTVKKEFRTRLVANNGLNPVYNEDPF **** ** * J ** • ***************** .*** .* * . . . * ** * * ********** .**

suPLCP VFRKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSLPTVFCC

Drosophila NorpA VFRKWLPDLAVLRFGVYEESGKILGQRILPLDGLQAGYRHVSLRTEANFPMSLPMLFW

**************. ^ ****, ** ; ;**************** . ***** ^ ***.*** .*

suPLCP IQLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIEENEISEVPTVNNKKRPTVR

Drosophila NorpA IELKXYVPDGFEDFMAMLSDPRGFAGAAKQQNEQMKALGIEEQSGGAARDAGKAKEEEKK

*: * * ****** ;*. ** * *. . g *... .*. .****. _ . * 89

suPLCP HPTTPTNTAPNIGGFTPGGPTHNQRMQPITAESLRKERAYQKLLKKQQKEQDSLSKRHSK Drosophila NorpA E P P LVFEPVTLESLRQEKGFQKVGKKQIKELDTLRKKHAK * * ***•*.*. • ** • ** * ** *• * *• *• *

suPLCP ERAMLQKTHYSTVNKMEACHDREKLTREKSLEKAIKKKGESNCSDLKIETEVHVQSLVND Drosophila NorpA ERTSVQKTQNAAIDKLIKGK SKD DIR NO ** • •* * * *• • ... *. . **

suPLCP HKARVKDWSDQSKEFADMVSRQQVEERSLKENHIQQRCDLLKKLMLSTQEEQMQLLAGR Drosophila NorpA --ANIKNSINDQTKQWTDMIARHRKEEWDMLRQHVQDSQDAMKALMLTVQAAQIKQLEDR * • * • • **.*. . .**. . ** . . * . * . * . * * * * . * * . • * *

suPLCP NDREMKELKSNQAKDSMESTKSVSHDKSIKNKAERDRRVRELQSNNTKKFLEERKR1.KMK Drosophila NorpA HARDIKDLNAKQAKMSADTAKEVQNDKTLKTKNEKDRRLREKRQNNVKRFMEEKKQIGVK *. . .*** * . • » * * . * * . . * * *.***•** • ** *.*.**•*. . . *

suPLCP QDEEKEEVQQEH Drosophila NorpA QGRAMEKLKLAHSKQIEEFSTDVQKLMDMYKIEEEAYKTQGKTEFCA 90

suPLCP MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEG

Rat PLCPl MAGAQPGVHALQLKPVCVSDSLKKGTKFVKWDD-DS-TIVTPIILRTDPQGFFFYWTDQN • . ** * • ** . * • * • • • * ** • % ... * ***.*.

suPLCP REGQVLELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYM

Rat PLCpl KETELLDLSLVKDARCGKHAKAP-KDPKLRELLDVGNIGHLEQRMITWYGPDLVNISHL .* • .* • ** • .* * * * *• • * *..*.* **. * *. * * * • ,

suPLCP HMVASAPEIAQVWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTRT

Rat PLCPl NLVAFQEEVAKEWTNEVFSLATNLLAQNMSRDAFLEKAYTKLKLQVTPEGRIPLKNIYRL • .* * *. * . * ...... *. *. * . *. * . *. # ** *•.****. .*• *

suPLCP FASGKTEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDIGRN

Rat PLCPl FSADR--KRVETALEACSLPSSRNDSIPQEDFTPDVYRVFLNNLCPRPEIDNIFSEFGAK

.***.: *.* **. : : : :

suPLCp ETEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSSDGL

Rat PLCpl SKPYLTVDQMMDFINLKQRDPRLNEILYPPLKQEQVQVLIEKYEPNSSLAKKGQMSVDGF ***********; * .:*:* :* : ** . . ** •* **•

suPLCp CRYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQVLLA

Rat PLCPl MRYLSGEENGWSPEKLDLNEDMSQPLSHYFINSSHNTYLTAGQLAGNSSVEMYRQVLLS ** * .:**. * ; J * « * .**_***************** *. *.***********.

suPLCP GCRCVELDCWDGRSEDQEPXITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILSFENHC

Rat PLCPl GCRCVELDCWKGRTAEEEPVITHGFTMTTEISFKEVIEAIAECAFKTSPFPILLSFENHV ********** _ ** . ..**.****• .* *.* *..**.** . ** ** **. .******

suPLCP SKP-QQLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNKRLKPEVEQ

Rat PLCpl DSPKQQAKMAEYCRLIFGDALLMEPLEKYPLESGVPLPSPMDLMYKILVKNKKKSHKSSE * ** *.* ** ..*. ** * *. .**.. ****** * ***.***. . .

suPLCP RQLELMRLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIGSISGSS

Rat PLCPl G SGKKK LSEQASNTYSDSSSVFE PSSPGAGEADTES

suPLCP IAAEDEIKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNYAQPV

Rat PLCPl DDDDDDD DCKK SSMDEGTAGSEAMAT EEMSNLVNYIQPV

•*• ** ***. .. * .********

SUPLCP KFQGFDVAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSSNYM

Rat PLCgl KFESFETSKKRNKSFEMSSFVETKGLEQLTKSPVEFVEYNKMQLSRIYPKGTRVDSSNYM **; . Î ; ;** *.**** * ; * * . .***.*** *«*****.* * ******

suPLCP PQIFWNAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSESPV

Rat PLCPl PQLFWNAGCQMVALNFQTVDLAMQINMGMYEYNGKSGYRLKPEFMRRPDKHFDPFTEGIV

***************** . *: S* * ; * * .**** ** ***. ****** . ****.* *

suPLCP DGVIAATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLNPVYNEE

Rat PLCPl DGIVANTLSVKIISGQFLSDKKVGTYVEVDMFGLPVDTRRKAFKTKTSQGNAVNPVWEEE **..* * **. .*******************.*** ** *. .*.* * .***..**

BuPLCg PFVFRKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSLPTVF

Rat PLCPl PIVFKKWLPSLACLRIAAYEEGGKFIGHRILPVQAIRPGYHYICLRNERNQPLMLPAVF *.**.***** ** **** ******. ** . **** . . _ .. ** . . * ** * * ** **.** 91

suPLCP CCIQLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIBENE---ISEVPTVNNKK

Rat PLCpl VYIEVKDYVPDTYADVIEALSNPIRYVNLMEQRAKQLAALTLEDEEEVKKEADPGETSSE * • •* * * * * •• • •***•* * • ******* • • * • » * * . «

suPLCP RPTVRHPTTPTNTAPNIGGFTPGGP THNQ RMQPI TA

Rat PLCpi APSETRTTPAENGVNHTATLAPKPPSQAPHSQPAPGSVKAPAKTEDLIQSVLTEVEAQTI *: .*.* .: : . *

suPLCP ESLRKERAYQKLLKKQQKEQDSLSKRHSKERAMLQKTHYSTVNKMEACHDREKLTREKSL

Rat PLCPl EELKQQKSFVKLQKKHYKEMKDLVKRHHKKTTELIKEHTTKYNEIQNDYLRRRAALE KSA * * * * * * • * * * * * * * • • * * * « * • • • • * • - * * *

suPLCp EKAIKKKGE-SNCSDLKIETEVHVQSLVNDHKARVKDWSDQSKEFADMVSRQQVEERSL

Rat PLCPl KKDSKKKSE PS S PDHGSSAIEQDLAALDAEMTQKLIDLKDKQQQQLLNLRQEQYYSEKYQ • * * * * * * * • * • • • * • * * * •

suPLCP KENHIQQRCDLLKKLMLSTQEEQMQLLAGRNDREMKELKSNQ-AKDSMESTKSVSHDKS-

Rat PLCPl KREHIKLLIQKLTDVAEECQNNQLKKLKEICEKEKKELKKKMDKKRQEKITEAKSKDKSQ

* J ** J ; * • •* ** * * E S . *. . *.** *

suPLCp x KNKAERDRRVRELQSNNTKKFLEERKRLKMKQDEEKE EVQQEH

Rat PLCpl MEEEKTEMIRSYIQEWQYIKRLEEAQSKRQEKLVEKHKEIRQQILDEKPKLQMELEQEY

* i ; T J ;* ; .* ** ; S : ;** *. .**.

suPLCP

Rat PLCPl QDKFKRLPLEILEFVQEAMKGKVSEDSNHGSAPPSLASDPAKVNLKSPSSEEVQGENAGR

suPLCP

Rat PLCPl EFDTPL 92

suPLCp MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEGREGQV

Rat PLCP2 MSLLNPVLLPPKVKAYLSQGERFIKWDDETS--IASPVILRVDPKGYYLYWTHQSKEMEF *. * *•* *•••* * •

SuPLCP LELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMHMVAS

Rat PLCP2 LDVTSIRDTRFGKFAKIPKSQKLREVFNMDFPDNHFLLKTFTWSGPDMVGLTFHNFVSY *...**** * . * .. .*.*.***•*••••*.

SUPLCP APEIAQVWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTRTFASGK Rat PLCP2 KENVGKDWAEDVLALAKHPMTANASRSTFLDKILVKLKMQLSPEGKIPVKNFFQMFPADR.. . * . .* • * * * .*• • * • *.*«***•*• • * .

suPLCP TEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDIGRNETEGL

Rat PLCP2 --KRVEAALSACHLAKGKNDAINPEDFPESVYKSFLMSLCPRPEIDEIFTSYHAKAKPYM ** i*. * ** * *. * *** . . : . ;

suPLCP SVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSSDGLCRYLM

Rat PLCP2 TKEHLTKFINQKQRDPRLNSLLFPPARPEQVQALIDKYEPSGINVQRGQLSPEGMVWFLC *. * .* - * . ******** .*** *.* .** ** ... ** .*. .*

suPLCP SDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQVLLAGCRCV

Rat PLCp2 GPENSVLAHDTLRIHQDMTQPLNHYFINSSHNTYLTAGQFSGPSSAEMYRQVLLSGCRCV . **; ; * *.;;*** *** _ ************* _ ** * **********.*****

suPLCP ELDCWDGRSEDQEPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILS FENHCSKP-Q

Rat PLCp2 ELDCWKGKPPDEEP11THGFTMTTDILFKEAVEAIAESAFKTSPYPVILSFENHVDSPRQ ******. *.*******..* ** * :;** ** ;********* * *

suPLCP QLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPS PNKLKRKILIKNKRLKPEVEQRQLEL

Rat PLCp2 QAKMAEYCRTMFGETLLTEPLENFPLKPGMPLPSPEDLRGKILIKNK * *. * ** ..** ** * * *. . ** ^ ^ .***** . *. *******

suPLCp MRLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPS KDSIGSISGSSIAAED

Rat PLCp2 KNQ FSGPASPNKKPDGVSEGGFPSSVPVEEDTGWTAEDRT ** .*..** :.*. .: ***.

suPLCP EIKLKHNMSVKMNAKKGE--LLSQEDEAAWAQYRYTGATTNIHACLSRLVNYAQPVKFQ

Rat PLCP2 EVEEGEEEEEVEEEEEEESGNLDEEEIKKMQSDEGTAGLEVTAYEEMSSLVNYIQPTKFI *: : .: . : :: * : :: :* .. : :* **** **.**

suPLCP GFDVAEERNLHFNMSSFNESVGLGYIKTQCIE FVNYNKRQMSRIYPQGRRYDSSNYMPQI

Rat PLCp2 SFEFSAQKNRSYLVSSFTELKAYELLSKASMQFVDYNKRQMSRVYPKGTRMDSSNYMPQM

„ *; . ; j i * ; .* * * * e Î ;**;********;**;* * ********.

suPLCP FWNAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSESPVDGV

Rat PLCP2 FWNAGCQMVALNFQTMDLPMQQNMALFEFNGQSGYLLKHEFMRRQDKQFNPFSVDRIDW

**************. *. ** * e **.** ** * * * .**** *. *.*** < .* *

suPLCP IAATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLNPVYNEEPFV

Rat PLCP2 VATTLSITVISGQFLSERSVRTYVEVELFGLPGDPKRRYRTKLSPTANSINPVWKEEPFI .*.* *. ********.. * *****...*** * ** . ^ .***..****.

SuPLCp FRKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSLPTVFCCI

Rat PLCP2 FEKILVPELASLRIAVMEEGGKFIGHRIIPINALHSGYHHLCLRSESNMPLTMPALFVFL

** * * * *****;**.**.*. . :**:*; e**«* *•**..*••* 93

suPLCP QLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMG--IEENEISEVPTVNNKKRPTV

Rat PLCp2 EMKDYVPDTWADLTVALANPIKYFSAHDKKSVKLKEVTGSLPEKLFSGIPVASQSNGAPV

:: * **** ** : : * *: * :* : : :* : : *: :* ..*

suPLCp RHPTTPTNTAPNIGGFTPGGPTHNQRMQPITAESLRKERAYQKLLKKQQKEQDSLSKRHS

Rat PLCP2 SAGNG- -STAPGTKAKEEAT-KEVAEPQTTSLEELRELKGWKLQRRHEKELRELERRGA

** * ^ e > e * • * ** . . ** * ; * •

suPLCP K-ERAMLQK- - - THYS TVNKME ACHDRE - KLTRE K SLEKAIKKKGESNCSD

Rat PLCP2 RRWEELLQRGAAQLAELQDPAASCKLRPGKGSRKKRIVPCEETIWPREVLEGPDPRVQD

: . : : * : * * : * : * : : : :

suPLCP LKIETEVHVQS LVNDHKARVKDWSDQSKEFADMVS-RQQVEERSLKENHIQQRCD

Rat PLCp2 LKDRLEQELQQQGEEQYRSVLKRKEQHVTEQIAKMMELAREKQAAELKSFKETSETDTKE

** . * .: *. .. * :: *: : * :: :* .* :*:**. : :

suPLCP LLKKLMLSTQEEQMQLLAGRND REMKELKSNQAKDSMESTKSVSHDKSIKNKAER

Rat PLCP2 MKKKLEAKRLERIQAMTKVTTDKVAQERLKREINNSHIQEWQAVKQMTETLERHQEKLE

• ** * < * . * * . * . . . > > . . . * • . . . .

suPLCP DR RVRELQSNNTKKFLEERK-RLKMKQDEEKEEVQQEH

Rat PLCP2 EKQTACLEQIQAMEKQFQEKALAEYEAKMKGLEAEVKESMRACFKACFPTEAEEKPERPC

: : : * * * : : : *

suPLCP

Rat PLC#2 EASEESCPQEPLVNKTDTQESRL 94

Rat PLCP3 MAGARPGVHALQLEPPTWETLRRGSKFIKWDEEASS--RNLVTLRVDPNGFFLYWTGPN

suPLCP --MAKQYVFQWQVCVP---EPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEG .* * ***•*•

Rat PLCP3 MEVDTLDISSIRDTRTGRYARLPKDP-KlREVLGFGGPDTR--LEEKLMTWAGPDPVNT

suPLCP REGQVLELSQINDVRKG---IAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNI *. *..***** *** ..* • * *

Rat PLCP3 TFLNFMAVQDDTVKVWSEELFKLAMNILAQNASRNTFLRKAYTKLKLQVNQDGRIPVKNI

suPLCP NYMHMVASAPEIAQVWFDGLLTITHNVKAMNVCPMQSLKKHWMKICFQVNPNGRIPVRNL .* * * .*****•*•

Rat PLCp3 LKMFSADK--KRVETALESCGLNFNRSESIRPDEFSLEIFERFLNKLCLRPDIDKILLEI

SUPLCP TRTFASGKTEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDI *. . * * * .* . ** * * .* .* * .*.* * ** • •• ;*

Rat PLCP3 GAKGKPYLTLEQLMDFINQKQRDPRLNEVLYPPLRSSQARLLIEKYEPNKQFLERDQMSM

suPLCP GRNETEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSS * . *• .* . * • .A.*.*********.*.* • •* : * * . ;*

Rat PLCP3 EGFSRYLGGEENGILPLEALDLSMDMTQPLSAYFINSSHNTYLTAGQLAGTSSVEMYRQA

suPLCP DGLCRYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQV .* • ** * .** *. *. * ** ** * * ************ *• * ********

Rat PLCP3 LLWGCRCVELDVWKGRPPEEEPFITHGFTMTTEVPLRDVLEAIAETAFKTSPYPVILSFE

suPLCP LLAGCRCVELDCWDGRSEDQEPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILSFE ** ******** * ** • •**.****. .* *•• •***»•** •*** ** •*******

Rat PLCP3 NHVDSAKQQAKMAEYCRSIFGEALLIDPLDKYPLSAGTPLPS PQDLMGRX LVKNKKRHRP

suPLCP NHCS-KPQQLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNK-RLKP ** ** *•* ** -.** ** . ** .** * *****. * .**.*** * •*

Rat_PLCb-3 STGVP-DSSVRKRPLEQSNSALSESSAATEPSSPQLGSP--SSDSCPGLSNGEEVGLEKT

suPLCP EVEQRQLELMRLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIGSI

Rat PLCP3 SLEPQKSLGEEGLNRGPNVLMPDRDREDEEEDEEEEETTDPKKPTTDEGTASSEVNATEE

suPLCP SGSSIAAEDEIKLKHNMSVKMNAKKGELLSQ -EDEAAWAQYR YTGATTNIHAC

Rat PLCP3 MSTLVNYVEPVKFKSFEAARKRNKCFEMSSFVETKAMEQLTKSPMEFVEYNKQQLSRXYP

suPLCP LSRLVNYAQPVKFQGFDVAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYP - * ** * * •****• *. * -** ****** *< .***.***.*.*****

Rat PLCP3 KGTRVDSSNYMPQLFWNVGCQLVALNFQTLDLPMQLNAGVFEYNGRSGYLLKPEFMRRPD

L suPLCfi QGRRYDSSNYMPQIFWNAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPD •* * ************ ***.******. *• **** * ***** ******. ******

Rat PLCP3 KSFDPFTEVIVDGIVANALRVKVISGQFLSDRKVGIYVEVDMFGLPVDT-RRKYRTRTSQ

suPLCP RTFDPFSESPVDGVIAATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVP •****•* ***.•* *. ********* . * * * ******. *** ** **..****

Rat PLCp3 GNSFNPVWDEEPFDFPKWLPTLASLRIAAFEEGGRFVGHRILPVSAIRSGYHYVCLRNE

suPLCP NNGLNPVYNEEPFVFRKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTE * •***. .* * * * * ***** ** **** .****. ..*.****. ...**... ** * 95

Rat PLCp3 ANQPLCLPALLIYTEASDYIPDDHQDYAEALINPIKHVSLMDQRAKQLAALIGESEAQAS

SuPLCp GNFPLSLPTVFCCIQLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIE-ENEIS * ** ** . . . . *« ** • • •* * .* • •* ..***** . ** • *

Rat PLC(i3 TEMCQETPSQQQGSQLSSNPAPNPLDASPRWPPGPTTSPTSTSLSSPGQRDDLIASILSE suPLCP EVPTVN NKKRPTVRHPTTPTNTAPNIGGFTPGG P- THNQR :** :

Rat PLCp3 VTPTPLEELRSHKAMVKLRSRQDRDLRELHKKHQRKAVALTRRLLDGLAQARAEGKCRPS suPLCP MQPITAESLRKERAYQKLLKKQQKEQDSLSKRHSKERAMLQKTHYSTVNKMEACHD-R-- : * . *.**..:* ** . * : . : : . *

Rat PLCP3 SSALSRATNVEDVKEEEKEAARQYREFQNRQVQSLLELREAQADAETERRLEHLKQAQQR

SUPLCP E-KLTREKSLEKAIK--K--KG---ESNCSDLKIETEVHVQSLVNDHKARVKDWSDQSK * *: ::: *:: .

Rat PLCP3 LREWLDAHTTQFKRLKELNEREKKELQKILDRKRMNSISEAKTREKHKKEVELTEINRR

suPLCP EFADMVSRQQVEERSLKENHIQQRCDLLKKLMLSTQEEQMQLLAGRNDREMKELKSNQAK

: *** : :: : ;*** . ; ; **.. : :

Rat PLCp3 HITESVNSIRRLEEAQ--KQRHERLLAGQQQVLQQLVEEEPKLVAQLTQECQEQRERLPQ SUPLCP DSMESTKSVSHDKSIKNKAERDRRVRELQSNNTKKFLEERK. .* *. *. ....** RLKMKQDEEKEEVQQ.*

Rat PLCP3 EIRRCLLGETSEGLGDGPLVACASNGHAAGSGGHQSGADSESQEENTQL

suPLCP EH 96

SUPLCP MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEGREGQV

Rat PLCP4 MAKPYEFNWQKEVPSFLQEGAVFDRYEE-ESFVFEPNCLFKVDBFGFFLTWKSEGKEGQV * * * * *.** ** *••**.*****• •* * ******* • *** • .****.****

suPLCp LELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMHMVAS

Rat PLCP4 LECSLINSIRLAAIPKDPKIliAALESVGKSE--NDLEGRILCVCSGTDLVNIGFTYMVAE ***** •* *** * * • *• ..** • .****.*** . .***

SUPLCP APEIAQVWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTRTFASGK

Rat PLCP4 NPEITKQWVEGLRSIIHNFRANNVS PMTCLKKHWMKLAFLTNTSGKIPVRSITRTFASGK ***.. * . * * . * * * .****** *******. * * *.**** ; ********

suPLCP TEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDIGRNETEGL

Rat PLCp4 TEKVIFQALKELGLPSGKNDEIEPAAFTYEKFYELTQKICPRTDIEDLFKKINGDKTDYL ***..*..* ******** ****** * . ****** . ********* * * * * ..*. *

suPLCP SVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELLSSDGLCRYLM

Rat PLCP4 TVDQLVSFLNEHQRDPRLNEILFPFYDAKRAMQIIEMYEPDEELKKKGLISSDGFCRYLM .*..*. ** * . . ************ . ** .* .***. ** * ** *.****.*****

suPLCP SDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQVLLAGCRCV

Rat PLCP4 SDENAPVFLDRLELYQEMDHPLAHYFISSSHNTYLTGRQFGGKSSVEMYRQVLLAGCRCV **************** • * . . * * • * * * * ^ ********************************

suPLCp ELDCWDGRSEDQEPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILSFENHCSKPQQ

Rat PLCP4 ELDCWDGKGEDQEPIITHGKAMCTDILFKDVIQAIKETAFVTSEYPVILSFENHCSKYQQ ******* J ********** ****** *.******. .****** . • ************ **

suPLCP LKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNKRLKPEVEQRQLELM

Rat PLCP4 YQMSKYCEDLFGDLLLKQALESHPLEPGRLLPSPNDLKRKILIKNKRLKPEVEKKQLEAL . . . . * * * . . . * . . * * . * . * * * . e _ ***** ***************** . -*** .

suPLCP RLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIGSISGSSIAAEDE

Rat PLCP4 KSMMEAGESAAPASILEDDNEEEIESAADQ-EEEAHPEYKFGNELSADDFS : * * . :.:

suPLCP IKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNYAQPVKFQGFD

Rat PLCp4 HKEAVANSVKKG-LVTVEDEQAWMASYKYVGATTNIHPYLSTMINYAQPVKFQGFH

* J J* ^ S ** * *. . ** * * .* *.* ******* ** . ************

suPLCP VAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDSSNYMPQIFWN

Rat PLCP4 VAEERNIHYNMSSFNESVGLGYLKTHAIEFVNYNKRQMSRIYPKGGRVDSSNYMPQIFWN ****** . * . ************* .**. **************** . * * ************

suPLCP AGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFSESPVDGVIAA

Rat PLCP4 AGCQMVSLNYQTPDLAMQLNQGKFEYNGSCGYLLKPDFMRRPDRTFDPFSETPVDGVIAA ****** .** • * .** . ************ ********************** . ********

suPLCp TCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLNPVYNEEPFVFRK

Rat PLCP4 TCSVQVISGQFLSDKKIGTYVEVDMYGLPTDTIRKEFRTRMVMNNGLNPVYNEESFVFRK **************** . ***************** . * * * * * *********** *****

suPLCP WLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSLPTVFCCIQLK

Rat PLCp4 VILPDLAVLRIAVYDDNNKLIGQRILPLDGLQAGYRHISLRNEGNKPLSLPTIFCNIVLK * - ************ . . _ *********************** *** ****** .** * * * 97

TYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIEENEISEVPTVN--NKKRP---TV TYVPDGLGDIVDALSDPKKFLSITEKRADQLRAMGIETSDIADVPSDTSKNDKKGKANPA ******* • . *******. .* * .* * * * * * * * * * .*..**. *.*:

RHPTTPTNTA---PNXGGFTPGG---PTHNQRMQPITAESLRKERAYQKLLKKQQKEQDS KANVTPQSSSELRPTTTAALGSGQEAKKGIELIPQVRIEDLKQMKAYLKHLKKQQKELNS : . ** *. .* : : : * . * : : : ** * ******* ; *

LSKRHSKERAMLQKTHYSTVNKMEACHDREKLTREKSLEKAIKKKGESNCSDLKIETEVH LKKKHAKEHSTMQKLHCTQVDKIVAQYDKEKSTHEKILEKAMKKKGGSNCLEIKKETEIK **.*.**.. » * * * . *.*• * •*•** * « * * ********* * * * ; ; * ***•»

VQSLVNDHKARVKDWSDQSKEFADMVSRQQVEERSLKENHIQQRCDLLKKLMLSTQEEQ IQTLTSDHKSKVKEIVAQHTKEWSEMINTHSAEEQEIRDLHLSQQCELLRKLLINAHEQQ . * • * ***..**..*.•••**...*. . **. ... * . *.*.**.**.. ..*.*

MQLLAGRNDREMKELKSNQAKDSMESTKSVSHDKSIKNKAERDRRVRELQSNNTKKFLEE TQQLKLSHDRESKEMRAHQAKISMENSKAISQDKSIKNKAERERRVRELNSSNTKKFLEE * * » * * * **• • • •*** * * * e . * - .* . ********** . ****** . * ********

RKRLKMKQD- --EEKEEVQQEH RKRLAMKQSKEMDQLKKVQLEHLEFLEKQNEQAKEMQQMVKLEAEMDRRPATW 98 suPLCP --MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEGRE- Sponge PLCP MAGHVSKRVLKQPDVQSILLSGVSFWKWEDGD--QVAQKAEFFADKDAHFLYAKPEDKSK * * . * .*. * ::**** .* * . *: suPLCp -GQVLELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNINYMH Sponge PLCP YNIIWDVTHISDVRAGKVPYKDSKLHEFLRSMTSSDYLEPEQFLTMVYRREGIVNLDHVQ suPLCP MVASAPEIAQVWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNLTP-TF Sponge PLCP LMAPSMDVAEKWKRAVNAIVFNMLRFNASTLTFFKRNYQRILLSCNGYGQIPVKSICRAL • • * » . . * . * • • * * . * . . * . . . . * . * *.***. . * • • suPLCP ASGKTEKIVFKSLDELGLPSGKMDE IDPADFPFEKFYELYHKICP--RTDIELLFK Sponge PLCP LLRVRDKTDMELFGIFDAQKKKMDDGTEYIQDTDFTWDKYEQLVNQLTKGRTSDMECVIK • * • » . • * * * • * . • * * . • * • . * • • • • * • * • • * suPLCP DIGRNE-TEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKEL Sponge PLCP DLGKSKKHQQLTLQEFHNFLTKCQRDPRLNTILYPPPTKQQAKELAEKYEG---KAVEDR

*1*1.1 ; ;** . „ ******* ** ;::;, * * J• suPLCp LSSDGLCRYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMY Sponge PLCp LTPRALLNFLMSEDNNVLGVENLDQFMDMTQPLSHYFINSSHNTYLTGHQLRSLSTVEMY * ; * .***• . * . . . * . • * * ****************** . * . _ *.**** suPLCP RQVLLAGCRCVELDCWDGRSEDQEPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVIL Sponge PLCP RQCLLLGCRCVELDLWP EGEDIIITHGGTLCTKIPFKDWEAISEYAFITSEFPVIL ** ** ******** * * .. ***** ..** * *.**..** . **.**.***** suPLCP SFENHCS-KPQ-QLKLARYCEEVLGE FLLTEKLDDHPLDASVPLPSPNKLKRKILIKNKR Sponge PLCP SIENHCRHKPLLIQKIASTFALVFGDKLQKSPLDDYPLEPGLPLPSPERLKGKILIKDKV *.**** * * *•* *.*. * ***.**. .*****. .** ******* suPLCP LKPEVEQRQ Sponge PLCP KKSKGIESSTGTLPRGAHLTSEDKAETLSRKSGTGDKVSDGNGGQATPTSKEFVMPSVAL *.*. .* : suPLCP LELMRLRPELLEVN QEDEVEECES GY Sponge PLCP TSLSASSLWKDKMDIANLREIVPLPESPLETSPGPRKTSPGLTETCPGSPGPIKTSPGP suPLCP SDGAVS PPGN TDAHPKFKFPSKDS Sponge PLCP VDTSPSSPLHWPTCTWSGLTTPSSANAGGPPGTSTELPLPESTPPPPLPPPSETTPFNL * ; * * * * e suPLCP IGSISGSS- IAAEDEIKLK HNMSVKMN Sponge PLCP ETVPIPGTPPPSSGVNRTEEIQDNESCPLSPLDTSTAPDPNIPTDSPKETAPHPVTPVNS :**: : * :: suPLCP AKKGELLSQ EDEAAWAQ YRYTGATTN IHACLSRLVNYA Sponge PLCP LKETAFQNRSLKKTFSGMTEDSMESTPRKEEEFVYNMTPARPSEGEAFVTGPLVDIINYC *: : .: **. .. : *.**.. : . * :: . suPLCP QPVKFQGFDVAEERNLHFNMS S FNESVGLGYIKTQCIEFVNYNKRQMS RIYPQGRRYDS S Sponge PLCP SGITFEGFEAAEKTNCSFYQSSFNEDKGVSLVKVTAREFVKYNMRQMSRIYPQGGRINSS . „*:**; . * *: * * * * * * * e * ; .* e 4 ***.** ********** * .* * 99

suPLCP NYMPQIFWNAGCQMVALNFQS PDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRT---FDP Sponge PLCP NYMPQIFWNVGCQLVALNFQSTDLPIQLNNAKYEFNGVTGYLLKPAAYLKERTTGGLFNP ********* f * * * . ******* # * . •***. *.*.** ****** ; * *.*

suPLCP FSESPVDGVIAATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLN Sponge PLCp FIQNPVENIVPATLTLKVISGIFLTEKRTNCLVEAQMFGLPSDTVN--HKFYTTKKEAPH * : : -.**** ** . :*:***:**: . *: *. :

suPLCP PVYNEEPFVFRKWLPDLAVliRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPL Sponge PLCP PWDHEPFTFAKILLPEMAMLRVAVYDEDRELLGQRSLPVNAIRAGYRHIPLRDKYNQPL **.. * * * * *••**..*•**.***.* .*.*** **.. . . ****** * * . * * *

suPLCp SLPTVFCCIQLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIEENEISEVPTVN Sponge PLCP SLASLFVHIMVDDWVGSDMEAIIAGLMDPIAFVN- S ALAELAI GTE -NEDQDPSLLS * • * * **

suPLCP -NKKRPTVRHPTTPTN---TAPNIGGFTPGGPTHNQRMQPITAESLR-KERAYQKLLKKQ Sponge PLCP GSSTLPRTRKPENTDARKDSTTTVASPFPPSASFDQKVDAATIPSSPPVGSTAAAASSMA * * • *

suPLCP QKEQDSLSKRHSKERAMLQKTHYSTVNKMEACHDREKLTREKSLEKAIKKKGESNCSDLK Sponge PLCP APETKSLTLNYPKAKVDDVLPIVQMVTÎCRPPPVAAQHVDDCXAFHESILKIEKKMVTDRM

suPLCP XETEVHVQSLVNDHKARVKDWSDQSKEFADMVSRQQVEERSLKENHIQQRCDLLKKLML Sponge PLCP KVXiKKHEKHLSDHGS KQS KGKQEEVASRIAALKDDHS KELLALDKKLLHERNVKLETLLN

suPLCP STQEEQMQLLAGRNDREMKELKSNQAKDSMESTKSVSHDKSIKNKAERDRRVRELQSNNT Sponge PLCP EAHKKEMKDLIAQHRSEINSVSS---ELIMFLKRRDSDTKGRMAFDERTXAVGTKNKGEL

suPLCP KKFLE-ERKRLKMKQDEEKEEVQQEH Sponge PLCP EKLHQKEKENLEKSQKEAKERLQKEYEESLNKLEHPKGTSGSPEQTKEEATAM : * : : . * . * **.:*:*: 100

suPLCP MAKQYVFQWQVCVPEPLKNGAMFDRYEDGDSGSVEQS CLFKVDEYGFFIYYKSE

Squid PLCP MAGVETAVRQIELKWPN-VPEQLIKGDKFLKWEEGSSS--FTEILLRVDPKGYFLYWKIE ; * *** * •* * : . * • ;* * *•*.*.* *

SuPLCp GRE-GQVLELSQINDVRKGIAPKDVGYLAKVGSIPDPFGRG--NIEDRTITICSGLDFVN

Squid PLCP GKEDSQLLDLAYLRDIRCAKYAKPP-KDKKVKDAGTNFGSSNIPLQDKCVTICHGYNYID

*; ;. * : * , .* ** , ** . :: * : ;* * * * ::::

suPLCP INYMHMVASAPEIAQVWFDGLLTITHNVKANNVCPMQSLKKHWMKI CFQVNPNGRI PVRN

Squid PLCP LDWIHLVAENSSVAAKWAEEVFSYAYNLLSLNKNQLGEWEKLYFRLTTVEMEKNKIPVKS :: : :*:**. .. : * * : :: : :: * : : * : . :* :: : :

suPLCP LTRTFASGKT-EKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFK

Squid PLCP IQKCLSKDKDDRTRVAKAIEKIGWPSGKNDAIEIKAFDFDTFFKFYLSLLERSEIEGIFK : : .. * *:::::* **** * *: * . : *::** :**

suPLCP DIGRNETEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQ--MIKKE

Squid PLCP ELSQN-KGNITTVMFRDFLNDMQRHPSLHKTLFPLYTDSQCDALINEYESAVNKKGKKKG :: . :* . : :**** **.* *: : ***: * . : :* : ** . : **

SUPLCP LLSSDGLCRYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEM

Squid PLCP QLTKEGLLYFLMCEENNLTPMHRLDLGANMKLSLAAYYINSSHNTYLTGHQLTGKSSVEI

*: . : * * ;* * „ ;* * . . ** . * ;* J *J *.***********.*. ******.

suPLCP YRQVLLAGCRCVELDCWDGRSEDQEPIITHGLAMCTDIQFRDVIQAIRDTAFVTSDFPVI

Squid PLCP YRQVLLTGCRCLELDCWDGK--DGEPIITHGFTMCTEVQFKDWHAIAECAFKVSEYPVI ******. * * * * . ******* . * *******. .***. •**.**..** . ** *. . * * *

SUPLCP LSFENHCSKPQQLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKIIilKNKRL

Squid PLCp LSFENHCSVPQQKLLAQYCHEAFGELLLDNPIDGHPLKPGVPLPTPYDLRKKILIKNKKI ******** ** * **.** * .**.** . .* ** * ^ ^ ****.* *..*******..

suPLCP KPEVEQRQLELMRLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIG

Squid PLCP HKGAG D--DDELAGLTD EEKKKIEKEKKD : * :: :* :*

suPLCP SISGSSIAAEDEIKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLV

Squid PLCP —AG T AAKEAEAA—EE MSA IA7 :* : ** :. * *: :: **

suPLCP NYAQPVKFQGFDVAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRY

Squid PLCp NYIQPVHFTTFEQAQKKDRHYEMSSMVETQALNKLKDNPEDFVDYNKKQLTRIYPKGTRV

** ***;* *; ; ;*** . *. e *s .* . .**.***.*,.****.* *

suPLCP DSSNYMPQIFWNAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDP

Squid PLCP DSSNYVPQIYWNAGCQLVALNFQCFDVAMCVNLGVFEYNGCSGYLLKPEFMRKLDKRFDP

*****. *** . ****** . ****** ^ * • e * • * * ****** ^ ******. *** . *. ** *

suPLCP FSES PVDGVIAATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLN

Squid PLCP FTESTVDGWAGTIEIKlISAQFLSDKQISSYVEVEMYGLPTDTVRKKFKTKTVNNNGMD

*. ** e ****.* _ * .. . ** ^ ******. . ^ .****.********.*.. .*;** ***..

suPLCP PVYNEEPFVFRKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPL

Squid PLCP PYYNENAFVFKKWLPDLAWRIIVNEDGGKFIGHRLMPLDGIKPGYRHIPLRNESNRPL

* ***;#***;************ * *.***.**.*..****. . ***** ** * * ** 101

suPLCP SLPTVFCCIQLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIEE NEISEV

Squid PLCP GLASVFAHIVAKDYVSDAFADFADALLNPIAYQSAQDARAAALCAFEDDPDAALNAAKPV * .** * **** .».**** .* ** . ** *

suPLCP PTVNNKKRPTVRHPTTPTNTAPNIGG FTPGGP TH

Squid PLCP KGKSPKAGKKAWAGAGKKVSQGNIAKSAFGGAKKAAAAGKAATPASPPPPPAAADSEFVQ *»* ** * ***

suPLCP NQRMQP ITAESLRKERAYQKLLKKQQKEQDSLSKRHSKERAMLQKTHYSTVNKM

Squid PLCP NSKMKEESAVALPKLDALKSNKAYAKIIS KRDKEVDSIKKKNDKAQSNMKTKMKAEEKKM •** *••** **• *

suPLCP EACHDREKLTREKS---LEKAIKKKGESNCSDLKIETEVHVQSLVNDHKARVKDWSDQS

Squid PLCP KDSHS KAEKKAEKSGKEQHDQLVKENQDKFNQLHKENIDKYLATCSEHFNAESEICKKYI

SUPLCP KEFADMVS RQQVEERSLKENHIQQRCDLLKKLMLSTQEEQMQLLAGRNDRE-MKE

Squid PLCP DSLNDCLKGVITSTQEDLKKKLQTIHDKEVESMNKELNKKSKEEQKNLGKETKDKEELAR *• *• .*.* .•***.* •*•*•

suPLCP LKSNQAKDSMESTKS-VSHDKSIKNKAE--RDRRVRELQSNNTKKFLEERKRLKMKQDEE

Squid PLCp KKRELSKKLIDGIVAERNKLKALKERQEKVLDKNVEELTKEYEKEKANAENKLKEDIDAK

suPLCP KEEVQQEH-

Squid PLCP LKKVKEEWS 102

suPLCP --MAKQYVFQWQVC---VPEPLKNGAMFDRYEDGDSGSVEQSCLFKVDEYGFFIYYKSEG

Xenopus PLCP MAGARPGVHSLQLEPVKVPETLIKGSKFIKWDE-ES-SSKSLVTLRVDTMGFYLYWTCPN * . * * . * * * * » * • * . * * * * . . * .

suPLCP REGQVLELSQINDVRKG---IAPKDVGYLAKVGSIPDPFGRGNIEDRTITICSGLDFVNI

Xenopus PLCP MEVDILDISVIRDTRTGKYAKIPKDIKMREILG-FTGPEQRP--EDKLVTWYGNDIVNI * ..*..* * * * * * * * * • • * - * * • ** *

suPLCP NYMHMVASAPEIAQVWFDGLLTITHNVKANNVCPMQSLKKHWMKICFQVNPNGRIPVRNL Xenopus PLCP SFLNFMAVQEDTAKIWTEELFKLAHNILAQNSSRNTFLQKAYTKLKLQVNQDGRIPVKNI *. • * • * » • .* * . * . * *- * • *• • ** * .*****•*•

suPLCP TRTFASGKTEKIVFKSLDELGLPSGKMDEIDPADFPFEKFYELYHKICPRTDIELLFKDI

Xenopus PLCP LKMFAADK--KRVETALESCGLNFNRGDSIKPEEFTLDIFERFLNKLCLRPDIDKILLEL * * • * * * • * • * * .* . * * **•

SuPLCp GRNETEGLSVEKLIEFLNDKQRDPRLNEILFPMYDRERVLQIIDTYEKDPQMIKKELIiSS

Xenopus PLCP GAKGKPYLTLEQLMDFLNQKQRDPRLNEILFPPLKRDQVRQLIEKYEPNRQFLDRDQMSM *• •* • * • •* * * . ************* *..* *.*. ** . *.

suPLCP DGLCRYLMSDENAPVFLDRLELYQDMEQPLSHYFINSSHNTYLTGRQFGGKSSVEMYRQV

Xenopus PLCP EGFSRFLGGHENSIVPPEILDLSDDMNQPLSSYFINSSHNTYLTAGQLTGNSSVEMYRQV »*• *•* **• * « *•* •**•**** ************ * • * . *********

suPLCP LLAGCRCVELDCWDGRS EDQEP11THGLAMCTDIQFRDVIQAIRDTAFVTSDFPVILS FE

Xenopus PLCP LLTGCRCIELDCWKGRPQDEEPFITHGFTMTTEIPFKEVIEAIAESAFKTSPFPVILSFE ************* * * •*•**•****..* *•* *••**•** • . * * * * ********

suPLCP NHCSKP-QQLKLARYCEEVLGEFLLTEKLDDHPLDASVPLPSPNKLKRKILIKNK-RLKP

Xenopus PLCP NHVDSSKQQAKMAEYCRNIFGDALLIDPLEKYPLQPGVALPSPQELMGKILVKNKKRHKV * * * * * • * * * ...*. * * . * . .**, * ****..* ******* * *

suPLCP EVEQRQLELMRLRPELLEVNQEDEVEECESGYSDGAVSPPGNTDAHPKFKFPSKDSIGSI

Xenopus PLCP AVKGPEGSVKRRVSEQTSNTYSDSSSVCESSAT-G--LPP-SESADVSLTLSNGD * * * * * *

suPLCP SGSSIAAEDEIKLKHNMSVKMNAKKGELLSQEDEAAWAQYRYTGATTNIHACLSRLVNY Xenopus PLCP --EKIEEKPPKYTKPRKSIDHDAYSEEEEEEEPSDPKKSDEGTASSEVNATEEMSTLVNY * • * *• •* * »* •• • • * .* ** * *

suPLCp AQPVKFQGFDVAEERNLHFNMSSFNESVGLGYIKTQCIEFVNYNKRQMSRIYPQGRRYDS Xenopus PLCP VEPVKFKSFDAAKKRNKYYEMSSFVETKALEQLTKSPMEFVEYNKKQLSRIYPKGTRVDS .****. ** *.»** «..**** *. * .***.***.*.*****.* * ** suPLCp SNYMPQIFWNAGCQMVALNFQSPDIGMQLNQGKFEYNGCCGYLLKPDFMRRPDRTFDPFS

Xenopus PLCP SNYMPQLFWNAGCQMVALNFQTLDLPMQLNVGIFEYNRRSGYLLKPEFMCRTDKHFDPFT ****** . ************** . * . * * * * * * * * * ****** . * * * * . ****.

SUPLC# ESPVDGVIAATCSVQVISGQFLSDKKVGTYVEVDMYGLPTDTIRREHRTRTVPNNGLNPV

Xenopus PLCP ENIVDGIVANTVKIKIISGQFLSEKRVGIFVEVDMFGLPVDTKR-KFRTKTSQGNSFNPV * ***..* * •••*******.*.** • ***** . * * * * * * . **.* * .***

SUPLCP YNEEPFVFRKWLPDLAVLRIAVYEEGGKLIGQRILPLDGLQAGYRHISLRTEGNFPLSL Xenopus PLCP WDEEPFILPKWLPTLATLRIAVFEEGGKFVGHRILPVSAIRPGYHYICLRNELNQPVCL ..****•• ***** *******.*****..*,****. .. **..**** * *. * 103

suPLCP PTVFCCIQLKTYVPDGLTQFVDALSDPQLYLSQAEKRAKQLLCMGIEENE---ISEVPTV

Xenopus PLCP PALLVYTEVNDYIPDDHQGYANALTNPIKHISLMDQRAKQLAALMGE - NEQPFNKDKIDW

*: : : :: : *:**. :: * ;; ***** ,: * ** .:

suPLCP NNKKRPTVR--HPTTPTNTAPNIGGFTPGGP THNQRMQPITAESLRKERAYQKL

Xenopus PLCP DPISKPPIRSRDDTPEKIKIPIIPVPSPPAQRDDHIASVLTDIQAPSMEELKSQKSFEKL

: .*...** :* . : :*, :*.*:.:::::**

suPLCp LKKQQKEQDSLSKRHSKERAMLQKTHYSTVNKMEACHDREKLTREKSLEKAIKKKGESNC

Xenopus PLCP XCRQYRELRQLRRKHLRKVSSLCKEQSTWVTPLQSLRRRKPFGR »* • * * •• * S .. * * .. * ... .*. .*

suPLCP SDLKIETEVHVQSLVNDHKARVKDWSDQSKEFADMVSRQQVEERSLKENHIQQRCDLLK

Xenopus PLCP SHRG VSGADQDAEQKLMDMQRDQQKRLLELREIQHEQERKLKHSHLLQAVQKLQ *. *.. :* : :: *: **.*.: :: . *: :**.**.. * : * : *:

suPLCP KLMLSTQEEQMQLLAGRNDREMKELKSNQ---AKDSMESTKSVSHDKSIKNKAERDRRVR

Xenopus PLCP EVAISYHSTQLKKLKEINEKEKKELQKILDRKRHNSITEAKSRERQKKDVELTEINRRHI • • • * • * • • * * « • * ** * • • • * • • ** • . * • .* . **

suPLCP ELQSNNTKKFLEERKR LKMKQDEE -KEEVQQEH

Xenopus PLCp NESVSSIRRLEEAQKRRQEKLQTAHQETLQRIKDEEPKLQAQLDEACQAEFCQLPQEVRR

: . .. :: : * :** *: :*** * :. * :.

suPLCp

Xenopus PLCp YLQEDGWVGSGSGPPSSSHSSPPSITRSWGSESGEKDSLHSPGDSSDEATRL 104

REFERENCES

Aarhus, R., Dickey, D.M., Graef^ R.M., Gee, K.R., Walseth, T.F., and Lee, H.C., 1996. Activation and inactivation of Ca^ release by NAADP^. J. Biol. Chem. 271, 8513- 8516.

Allen, V., Swigart, P., Cheung, R., Cockcroft, S., and Katan, M., 1997. Regulation of inositol lipid-specific phospholipase cdelta by changes in Ca^ ion concentrations. Biochem. J. 327 (Pt 2), 545-552.

Bae, Y.S., Cantley, L.G., Chen, C.S., Kim, S.R., Kwon, K.S., and Rhee, S.G., 1998. Activation of phospholipase C-gamma by phosphatidylinositol 3,4,5- trisphosphate. J. Biol. Chem. 273, 4465-4469.

Barr, A.J., Ali, H., Haiibabu, B., Snyderman, R., and Smrcka, A V., 2000. Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits. Biochemistry. 39, 1800-1806.

Berridge, M.J., 1993. Inositol trisphosphate and calcium signalling. Nature. 361,315-325.

Boyer, J.L., Graber, S.G., Waldo, G.L., Harden, T.K., and Garrison, J.C., 1994. Selective activation of phospholipase C by recombinant G-protein alpha- and beta gamma- subunits. J. Biol. Chem. 269,2814-2819.

Buck, W.R., Rakow, T.L., and Shen, S.S., 1992. Synergistic release of calcium in sea urchin eggs by caffeine and ryanodine. Exp. Cell Res. 202, 59-66.

Buck, W.R., Hoffmann, E.E., Rakow, T.L., and Shen, S.S., 1994. Synergistic calcium release in the sea urchin egg by ryanodine and cyclic ADP ribose. Dev. Biol. 163,1-10.

Cameron, R.A., Mahairas, G., Rast, J.P., Martinez, P., Biondi, T.R., Swartzell, S., Wallace, J.C., Poustka, A.J., Livingston, B.T., Wray, G.A., Ettensohn, C.A., Lehrach, H., Britten, R.J., Davidson, E.H., and Hood, L., 2000. A sea urchin genome project: sequence scan, virtual map, and additional resources. Proc. Natl. Acad. Sci. U. S. A. 97, 9514-9518.

Carafbli, E., 1987. Intracellular calcium homeostasis. Annu. Rev. Biochem. 56,395-433.

Carroll, D.J., Ramarao, C.S., Mehlmann, L.M., Roche, S., Terasaki, M., and Jaffe, L.A., 1997. Calcium release at fertilization in starfish eggs is mediated by phospholipase Cgamma. J. Cell Biol. 138,1303-1311.

Carroll, D.J., Albay, D.T., Terasaki, M., Jaffe, L.A., and Foltz, K.R., 1999. Identiûcation of PLCgamma-dependent and -independent events during fertilization of sea urchin eggs. Dev. Biol. 206,232-247. 105

Chini, E.N., Beers, K.W., and Dousa, T.P., 1995. Nicotinate adenine dinucleodde phosphate (NAADP) triggers a specific calcium release system in sea urchin eggs. J. Biol. Chem. 270,3216-3223.

Chini, E.N. and De Toledo, F.G., 2002. Nicotinic acid adenine dinucleotide phosphate: a new intracellular second messenger? Am. J. Physiol Cell Physiol. 282, Cl 191-C1198.

Ciapa, B. and Whitaker, M., 1986. Two phases of inositol polyphosphate and diacylglycerol production at fertilisation. FEBS Lett. 195, 347-351.

Ciapa, B. and Epel, D., 1996. An early increase in cGMP follows fertilization of sea urchin eggs. Biochem. Biophys. Res. Commun. 223, 633-636.

Clapham, D.E., 1995. Calcium signaling. Cell. 80,259-268.

Clapper, D.L., Walseth, T.F., Dargie, P.J., and Lee, H.C., 1987. Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate. J. Biol. Chem. 262, 9561-9568.

Clementi, E., Riccio, M., Sciorati, C., Nistico, G., and Meldolesi, J., 1996. The type 2 ryanodine receptor of neurosecretory PC12 cells is activated by cyclic ADP-ribose. Role of the nitric oxide/cGMP pathway. J. Biol. Chem. 271, 17739-17745.

Coward, K., Owen, H., Poustka, A.J., Hibbitt, O., Tunwell, R., Kubota, H., Swann, K., and Parrington, J., 2004. Cloning of a novel phospholipase C-delta isofbrm from pacific purple sea urchin {Strongylocentrotus purpuratus) gametes and its expression during early embryonic development. Biochem. Biophys. Res. Commun. 313, 894-901.

Crossley, I., Whalley, T., and Whitaker, M., 1991. Guanosine 5'-thiotriphosphate may stimulate phosphoinositide messenger production in sea urchin eggs by a different route than the fertilizing sperm. Cell Regul. 2,121-133.

Currie, K.P., Swann, K., Galione, A., and Scott, R.H., 1992. Activation of Ca(2+)-dependent currents in cultured rat dorsal root ganglion neurones by a sperm factor and cyclic ADP-ribose. Mol. Biol. Cell. 3,1415-1425.

Dargie, P.J., Agre, M.C., and Lee, H.C., 1990. Comparison of Ca^ mobilizing activities of cyclic ADP-ribose and inositol trisphosphate. Cell Regul. 1,279-290.

De Nadai, C., Cailliau, K., Epel, D., and Ciapa, B., 1998. Detection of phospholipase Cgamma in sea urchin eggs. Dev. Growth Differ. 40, 669-676.

Dupont, G., McGuinness, O.M., Johnson, M.H., Berridge, M.J., and Borgese, F., 1996. Phospholipase C in mouse oocytes: characterization of beta and gamma isofbrms and their possible involvement in sperm-induced Ca^ spiking. Biochem. J. 316 (Pt 2), 583-591. 106

Ellis, M.V., James, S R., Perisic, O., Downes, C.P., Williams, R.L., and Katan, M., 1998. Catalytic domain of phosphoinositide-speciGc phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of plcdeltal. J. Biol. Chem. 273, 11650-11659.

Essen, L.O., Perisic, O., Cheung, R., Katan, M., and Williams, R.L., 1996. Crystal structure of a mammalian phosphoinositide-speciGc phospholipase C delta. Nature. 380, 595- 602.

Falasca, M., Logan, S.K., Lehto, V.P., Baccante, G., Lemmon, M.A., and Schlessinger, J., 1998. Activation of phospholipase C gamma by PI 3-kinase-induced PH domain- mediated membrane targeting. EMBO J. 17,414-422.

Feng, J.F., Rhee, S.G., and Im, M.J., 1996. Evidence that phosphohpase deltal is the effector in the Gh (transglutaminase Il)-mediated signaling. J. Biol. Chem. 271, 16451-16454.

Ferreira, P.A., Shortridge, R.D., and Pak, W.L., 1993. Distinctive subtypes of bovine phospholipase C that have preferential expression in the retina and high homology to the norpA gene product of Drosophila. Proc. Natl. Acad. Sci. U. S. A. 90, 6042-6046.

Fujino, Y., Mitsunaga, K., Fujiwara, A., and Yasumasu, I., 1985. Inhibition of ^Ca^ uptake in the eggs and embryos of the sea urchin, /dMf&ocw&zrza cra&?tspz/wz, by several calcium antagonists, anion transport inhibitor, and chloride transport inhibitors. J. Exp. Zool. 235,281-288.

Fujiwara, A., Sudoh, K., and Yasumasu, L, 1988. Activation of Sea Urchin Eggs by Halothane and its Inhibition by Dantrolene. Dev. Growth Differ. 30, 1-8.

Fukui, T., Lutz, R.J., and Lowenstein, J.M., 1988. Puriûcation of a phosphohpase C from rat liver cytosol that acts on phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate. J. Biol. Chem. 263,17730-17737.

Galione, A., White, A., Willmott, N., Turner, M., Potter, B.V., and Watson, S.P., 1993a. cGMP mobilizes intracellular Ca^ in sea urchin eggs by stimulating cyclic ADP- ribose synthesis. Nature. 365,456-459.

Galione, A., McDougall, A., Busa, W.B., Willmott, N., Gillot, I., and Whitaker, M., 1993b. Redundant mechanisms of calcium-induced calcium release underlying calcium waves during fertilization of sea urchin eggs. Science. 261,348-352.

Genazzani, A.A. and Galione, A., 1996. Nicotinic acid-adenine dinucleotide phosphate mobilizes Ca^ 6om a thapsigargin-insensitive pool. Biochem. J. 315 ( Pt 3), 721- 725.

Genazzani, A.A., Empson, R.M., and Galione, A., 1996. Unique inactivation properties of NAADP-sensitive Ca^ release. J. Biol. Chem. 271,11599-11602. 107

Genazzani, A.A. and Billington, R.A., 2002. NAADP: an atypical Ca(2+)-release messenger? Trends Pharmacol. Sci. 23, 165-167.

Gill, D.M., Cholera toxin catalyzed ADP-ribosylation of membrane proteins. In: Hayaishi,0. and UedaJK. (Eds.), ADP-Ribosylation Reactions Biology and Medicine, Academic Press, New York, 1982, pp. 593-621.

Glahn, D., Mark, S.D., Behr, R.K., and Nuccitelh, R., 1999. Tyrosine kinase inhibitors block sperm-induced egg activation in Aemqpwf Dev. Biol. 205,171-180.

Grobler, J.A. and Hurley, J.H., 1998. Catalysis by phosphohpase C deltal requires that Ca2+ bind to the catalytic domain, but not the C2 domain. Biochemistry. 37, 5020-5028.

Guo, X. and Becker, P.L., 1997. Cyclic ADP-ribose-gated Ca^ release in sea urchin eggs requires an elevated. J. Biol. Chem. 272, 16984-16989.

Hanafy, K.A., Krumenacker, J.S., and Murad, F., 2001. NO, nitrotyrosine, and cyclic GMP in signal transduction. Med. Sci. Monit. 7, 801-819.

Homma, Y., Imaki, J., Nakanishi, O., and Takenawa, T., 1988. Isolation and characterization of two different forms of inositol phospholipid-specific phospholipase C from rat brain. J. Biol. Chem. 263, 6592-6598.

Homma, Y. and Emori, Y., 1995. A dual functional signal mediator showing RhoGAP and phospholipase C- delta stimulating activities. EMBO J. 14, 286-291.

Hyslop, L.A., Carroll, M., Nixon, V.L., McDougall, A., and Jones, K.T., 2001. Simultaneous measurement of intracellular nitric oxide and free calcium levels in chordate eggs demonstrates that nitric oxide has no role at fertilization. Dev. Biol. 234,216-230.

Jaffe, L.F., 1991. The path of calcium in cytosolic calcium oscillations: a unifying hypothesis. Proc. Natl. Acad. Sci. U. S. A. 88, 9883-9887.

Jhon, D.Y., Lee, H.H., Park, D., Lee, C.W., Lee, K.H., Yoo, O.J., and Rhee, S.G., 1993. Cloning, sequencing, purification, and Gq-dependent activation of phosphohpase C- beta 3. J. Biol. Chem. 268,6654-6661.

Jiang, H., Wu, D., and Simon, M.I., 1994. Activation of phosphohpase C beta 4 by heterotrimeric GTP-binding proteins. J. Biol. Chem. 269, 7593-7596.

Jones, K.T., Matsuda, M., Parrington, J., Katan, M., and Swann, K,, 2000. Different Ca^- releasing abihties of sperm extracts compared with tissue extracts and phospholipase C isofbrms in sea urchin egg homogenate and mouse eggs. Biochem. J. 346 Pt 3,743- 749.

Katan, M., 1998. Families of phosphoinositide-specific phosphohpase C: structure and function. Biochim. Biophys. Acta. 1436, 5-17. 108

Kim, H., Jacobson, E.L., and Jacobson, M.K., 1993. Synthesis and degradation of cyclic ADP-ribose by NAD glycohydrolases. Science. 261, 1330-1333.

Kline, D., 1988. Calcium-dependent events at fertilization of the frog egg: injection of a calcium buffer blocks ion channel opening, exocytosis, and formation of pronuclei. Dev. Biol. 126,346-361.

Koshiyama, H., Lee, H.C., and Tashjian, A.H., Jr., 1991. Novel mechanism of intracellular calcium release in pituitary cells. J. Biol. Chem. 266,16985-16988.

Koyanagi, M., Ono, K,, Suga, H., Iwabe, N., and Miyata, T., 1998. Phosphohpase C cDNAs 6om sponge and hydra: antiquity of genes involved in the inositol phospholipid signaling pathway. FEBS Lett. 439,66-70.

Kozasa, T. and Oilman, A G., 1995. Purification of Recombinant G-Proteins 6om j/P Cells by Hexahistidine Tagging of Associated Subunits - Characterization of Alpha(12), and Inhibition of Adenylyl-Cyclase by Alpha(Z). Journal of Biological Chemistry. 270,1734-1741.

Kuang, Y., Wu, Y., Smrcka, A., Jiang, H., and Wu, D., 1996. Identification of a phosphohpase C beta2 region that interacts with Gbeta-gamma. Proc. Natl. Acad. Sci. U. S. A. 93,2964-2968.

Kuo, R.C., Baxter, G.T., Thompson, S.H., Strieker, S.A., Patton, C., Bonaventura, J., and Epel, D., 2000. NO is necessary and sufficient for egg activation at fertilization. Nature. 406,633-636.

Kuroda, R., Kontani, K., Kanda, Y., Katada, T., Nakano, T., Satoh, Y., Suzuki, N., and Kuroda, H., 2001. Increase of cGMP, cADP-ribose and inositol 1,4,5-trisphosphate preceding Ca(2+) transients in fertilization of sea urchin eggs. Development. 128, 4405-4414.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227,680-685.

Leckie, C., Empson, R., Becchetti, A., Thomas, J., Galione, A., and Whitaker, M., 2003. The NO pathway acts late during the fertilization response in sea urchin eggs. J. Biol. Chem 278,12247-12254.

Lee, C.W., Park, D.J., Lee, K.H., Kim, C.G., and Rhee, S.G., 1993. Purification, molecular cloning, and sequencing of phosphohpase C-beta 4. J. Biol. Chem. 268,21318-21327.

Lee, C.W., Lee, K.H., Lee, S B., Park, D., and Rhee, S.G., 1994. Regulation of phosphohpase C-beta 4 by ribonucleotides and the alpha subunit of Gq. J. Biol. Chem. 269,25335-25338. 109

Lee, H.C. and Aarhus, R., 1991. ADP-ribosyl cyclase: an enzyme that cyclizes NAD^ into a calcium- mobilizing metabolite. Cell Regul. 2,203-209.

Lee, H.C., 1993. Potentiation of calcium- and caffeine-induced calcium release by cyclic ADP-ribose. J. Biol. Chem. 268,293-299.

Lee, H.C. and Aarhus, R., 1995. A derivative of NADP mobilizes calcium stores insensitive to inositol trisphosphate and cychc ADP-ribose. J. Biol. Chem. 270,2152-2157.

Lee, H.C. and Aarhus, R., 2000. Functional visualization of the separate but interacting calcium stores sensitive to NAADP and cychc ADP-ribose. J. Cell Sci. 113 Pt 24, 4413-4420.

Lee, K.Y., Ryu, S.H., Suh, P.G., Choi, W.C., and Rhee, S.G., 1987. Phosphohpase C associated with particulate fractions of bovine brain. Proc. Natl. Acad. Sci. U. S. A. 84, 5540-5544.

Lee, S.J., Christenson, L., Martin, T., and Shen, S.S., 1996. The cychc GMP-mediated calcium release pathway in sea urchin eggs is not required for the rise in calcium during fertilization. Dev. Biol. 180,324-335.

Lee, S.J. and Shen, S.S., 1998. The calcium transient in sea urchin eggs during fertilization requires the production of inositol 1,4,5-trisphosphate. Dev. Biol. 193,195-208.

Lopez, I., Mak, B.C., Ding, J., Hamm, H.E., and Lomasney, J.W., 2001. A novel Afunctional phosphohpase c that is regulated by Galpha 12 and stimulates the Ras/mitogen- activated protein kinase pathway. J. Biol. Chem. 276,2758-2765.

Ma, H.W., Bhtzer, R.D., Healy, B.C., Premont, R.T., Landau, E.M., and Iyengar, R., 1993. Receptor-evoked CI" current in AewqpMg oocytes is mediated through a beta-type phosphohpase C. Cloning of a new form of the enzyme. J. Biol. Chem. 268, 19915- 19918.

McPherson, S.M., McPherson, P.S., Mathews, L., Campbell, K.P., and Longo, F.J., 1992. Cortical localization of a calcium release channel in sea urchin eggs. J. Cell Biol. 116, 1111-1121.

Mehlmann, L.M., Carpenter, G., Rhee, S.G., and Jaffe, L.A., 1998. SH2 domain-mediated activation of phosphohpase Cgamma is not required to initiate Ca^ release at fertilization of mouse eggs. Dev. Biol. 203,221-232.

Meszaros, L.G., Bak, J., and Chu, A., 1993. Cychc ADP-ribose as an endogenous regulator of the non-skeletal type ryanodine receptor Ca^ channel. Nature. 364, 76-79.

Min, D.S., Kim, D.M., Lee, Y.H., Seo, J., Suh, P.G., and Ryu, S.H., 1993. Purification of a novel phosphohpase C isozyme from bovine cerebellum. J. Biol. Chem. 268, 12207- 12212. 110

Mohri, T., Ivonnet, P.I., and Chambers, E.L., 1995. Efkct on sperm-induced activation current and increase of cytosolic Ca^ by agents that modify the mobilization of [Ca^]{. I. Heparin and pentosan polysulfate. Dev. Biol. 172,139-157.

Moore, K.L. and Kinsey, W.H., 1995. ESects of protein tyrosine kinase inhibitors on egg activation and fertilization-dependent protein tyrosine kinase activity. Dev. Biol. 168, 1-10.

Neer, E.J., 1995. Heterotrimeric G proteins: organizers of transmembrane signals. Cell. 80, 249-257.

Park, D., Jhon, D.Y., Lee, C.W., Ryu, S.H., and Rhee, S.G., 1993. Removal of the carboxyl- terminal region of phospholipase C-beta 1 by calpain abolishes activation by G alpha q. J. Biol. Chem. 268,3710-3714.

Parys, J.B., McPherson, S.M., Mathews, L., Campbell, K.P., and Longo, F.J., 1994. Presence of inositol 1,4,5-trisphosphate receptor, calreticulin, and calsequestrin in eggs of sea urchins and Xenopus laevis. Dev. Biol. 161, 466-476.

Patton, C., Thompson, S., and Epel, D., 2004. Some precautions in using chelators to buffer metals in biological solutions. Cell Calcium. 35,427-431.

Rakow, T.L. and Shen, S.S., 1994. Molecular cloning and characterization of protein kinase C from the sea urchin Zyfec&wzwa Dev. Growth Differ. 36,489-497.

Rameh, L.E., Rhee, S.G., Spokes, K., Kazlauskas, A., Cantley, L.C., and Cantley, L.G., 1998. Phosphoinositide 3-kinase regulates phospholipase Cgamma-mediated calcium signaling. J. Biol. Chem. 273,23750-23757.

Rebecchi, M.J. and Rosen, O.M., 1987. Purification of a phosphoinositide-speciGc phosphohpase C from bovine brain. J. Biol. Chem. 262,12526-12532.

Rebecchi, M.J., Ebeihardt, R,, Delaney, T., Ah, S., and Bittman, R., 1993. Hydrolysis of short acyl chain inositol hpids by phospholipase C-delta 1. J. Biol. Chem. 268, 1735- 1741.

Rebecchi, M.J. and Scarlata, S., 1998. Pleckstrin homology domains: a common fold with diverse functions. Annu. Rev. Biophys. Biomol. Struct. 27, 503-528.

Rebecchi, M.J. and Pentyala, S., 2001. Structure, Function, and Control of Phosphoinositide- SpeciSc Phosphohpase C. Physiological Reviews. 80,1291-1335.

Rhee, S.G. and Bae, Y.S., 1997. Regulation of phosphoinositide-specific phosphohpase C isozymes. J. Biol. Chem. 272,15045-15048.

Rhee, S.G., 2001. REGULATION OF PHOSPHOINOSITIDE-SPECIFIC PHOSPHOLIPASE C*. Annu. Rev. Biochem. 70,281-312. Ill

Rongish, B.J., Wu, W., and Kinsey, W.H., 1999. Fertilization-induced activation of phospholipase C in the sea urchin egg. Dev. Biol. 215,147-154.

Runft, L.L., Watras, J., and Jaffe, L.A., 1999. Calcium release at fertilization of Aemopit; eggs requires type I IP(3) receptors, but not SH2 domain-mediated activation of PLCgamma or G(q)- mediated activation of PLCbeta. Dev. Biol. 214,399-411.

Runnels, L.W. and Scarlata, S.F., 1998. Regulation of the rate and extent of phosphohpase C beta 2 effector activation by the beta gamma subunits of heterotrimeric G proteins. Biochemistry. 37, 15563-15574.

Rusinko, N. and Lee, H.C., 1989. Widespread occurrence in animal tissues of an enzyme catalyzing the conversion of NAD+ into a cyclic metabolite with intracellular Ca2+- mobilizing activity. J. Biol. Chem. 264, 11725-11731.

Ryu, S.H., Cho, K.S., Lee, K.Y., Suh, P.G., and Rhee, S.G., 1987. Purification and characterization of two immunologically distinct phosphoinositide-specific phospholipases C from bovine brain. J. Biol. Chem. 262, 12511-12518.

Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A.V., 1998. Identification of a structural element in phosphohpase C beta2 that interacts with G protein betagamma subunits. J. Biol. Chem. 273, 7148-7154.

Sato, K., Iwasaki, T., Tamaki, I., Aoto, M., Tokmakov, A.A., and Fukami, Y., 1998. Involvement of protein-tyrosine phosphorylation and dephosphorylation in sperm- induced Xenopus egg activation. FEBS Lett. 424, 113-118.

Saunders, C M., Larman, M.G., Parrington, J., Cox, L.J., Royse, J., Blayney, L.M., Swann, K., and Lai, F.A., 2002. PLC zeta: a sperm-speciGc trigger of Ca(2+) oscillations in eggs and embryo development. Development. 129, 3533-3544.

Schmidt, T., Patton, C., and Epel, D., 1982. Is there a role for the Ca^ influx during fertilization of the sea urchin egg? Dev. Biol. 90,284-290.

Shearer, J., De Nadai, C., Emily-Fenouil, F., Gâche, C., Whitaker, M., and Ciapa, B., 1999. Role of phosphohpase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos. Development. 126,2273-2284.

Shen, S.S., 1989. Na*-H* antiport during fertilization of the sea urchin egg is blocked by W-7 but is insensitive to K252a and H-7. Biochem. Biophys. Res. Commun. 161, 1100- 1108.

Shen, S.S. and Buck, W.R., 1993. Sources of calcium in sea urchin eggs during the fertilization response. Dev. Biol. 157, 157-169.

Shen, S.S., 1995. Mechanisms of calcium regulation in sea urchin eggs and their activities during fertilization. Curr. Top. Dev. Biol. 30,63-101. 112

Shen, S.S., Kinsey, W.H., and Lee, S.J., 1999. Protein tyrosine kinase-dependent release of intracellular calcium in the sea urchin egg. Dev. Growth Differ. 41,345-355.

Shilling, F.M., Carroll, D.J., Muslin, A.J., Escobedo, J.A., Williams, L.T., and Jaffe, L.A., 1994. Evidence for both tyrosine kinase and G-protein-coupled pathways leading to starfish egg activation. Dev. Biol. 162, 590-599.

Shiwa, M., Murayama, T., and Ogawa, Y., 2002. Molecular cloning and characterization of ryanodine receptor from unfertilized sea urchin eggs. Am. J. Physiol Regul. Integr. Comp Physiol. 282, R727-R737.

Smrcka, A.V. and Stemweis, P.C., 1993. Regulation of purified subtypes of phosphatidylinositol-specific phosphohpase C beta by G protein alpha and beta gamma subunits. J. Biol. Chem. 268,9667-9674.

Song, C., Hu, C.D., Masago, M., Kariyai, K., Yamawaki-Kataoka, Y., Shibatohge, M., Wu, D., Satoh, T., and Kataoka, T., 2001. Regulation of a novel human phosphohpase C, PLCepsilon, through membrane targeting by Ras. J. Biol. Chem. 276,2752-2757.

Steinhardt, R.A. and Epel, D., 1974. Activation of sea-urchin eggs by a calcium ionophore. Proc. Natl. Acad. Sci. U. S. A. 71,1915-1919.

Strieker, S.A., Centonze, V.E., and Melendez, R.F., 1994. Calcium dynamics during starfish oocyte maturation and fertilization. Dev. Biol. 166,34-58.

Strieker, S.A., 1999. Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211,157-176.

Swann, K., Larman, M.G., Saunders, C.M., and Lai, F.A., 2004. The cytosohc sperm factor that triggers Ca^ oscillations and egg activation in mammals is a novel phosphohpase C: PLCzeta. Reproduction. 127,431-439.

Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H., 1993. Cychc ADP-ribose in insulin secretion from pancreatic beta cells. Science. 259,370-373.

Touhara, K., Inglese, J., Pitcher, J.A., Shaw, G., and Lefkowitz, R.J., 1994. Binding of G protein beta gamma-subunits to pleckstrin homology domains. J. Biol. Chem. 269, 10217-10220.

Towbin, H., Staehelin, T., and Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U. S. A. 76,4350-4354.

Turner, PR, Sheetz, M.P., and JaSe, L.A., 1984. Fertilization increases the polyphosphoinositide content of sea urchin eggs. Nature. 310,414-415. 113

Turner, P R., JaSe, L.A., and Fein, A., 1986. 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.

Turner, P.R., Jaffe, L.A., and Primakof^ P., 1987. A cholera toxin-sensitive G-protein stimulates exocytosis in sea urchin eggs. Dev. Biol. 120,577-583.

Voronina, E. and Wessel, G.M., 2004. Regulatory contribution of heterotrimeric G-proteins to oocyte maturation in the sea urchin. Mech. Dev. 121,247-259.

Wada, K,, Aota, S., Tsuchiya, R., Ishibashi, F., Gqjobori, T., and Ikemura, T., 1990. Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Res. 18 Suppl, 2367-2411.

Wahl, M.I., Jones, G.A., Nishibe, S., Rhee, S.G., and Carpenter, G., 1992. Growth factor stimulation of phosphohpase C-gamma 1 activity. Comparative properties of control and activated enzymes. J. Biol. Chem. 267,10447-10456.

Walseth, T.F., Aarhus, R., Zeleznikar, R.J., Jr., and Lee, H.C., 1991. Determination of endogenous levels of cychc ADP-ribose in rat tissues. Biochim. Biophys. Acta. 1094, 113-120.

Wang, S., Gebre-Medhin, S., Betsholtz, C., Stalberg, P., Zhou, Y., Larsson, C., Weber, G., Feinstein, R., Oberg, K,, Gobi, A., and Skogseid, B., 1998. Targeted disruption of the mouse phosphohpase C beta3 gene results in early embryonic lethality. FEBS Lett. 441,261-265.

Wang, T., Pentyala, S., Rebecchi, M.J., and Scarlata, S., 1999a. Differential association of the pleckstrin homology domains of phosphohpases C-beta 1, C-beta 2, and C-delta 1 with hpid bilayers and the beta gamma subunits of heterotrimeric G proteins. Biochemistry. 38,1517-1524.

Wang, T., Pentyala, S., Elliott, J.T., Dowal, L., Gupta, E., Rebecchi, M.J., and Scarlata, S., 1999b. Selective interaction of the C2 domains of phospholipase C-betal and - beta2 with activated Galphaq subunits: an alternative function for C2 signaling modules. Proc. Natl. Acad. Sci. U. S. A. 96, 7843-7846.

Wang, T., Dowal, L., El Maghrabi, M.R., Rebecchi, M., and Scarlata, S., 2000. The pleckstrin homology domain of phosphohpase C-beta(2) links the binding of gbetagamma to activation of the catalytic core. J. Biol. Chem. 275,7466-7469.

Whalley, T., McDougall, A., Crossley, L, Swann, K., and Whitaker, M., 1992. Internal calcium release and activation of sea urchin eggs by cGMP are independent of the phosphoinositide signaling pathway. Mol. Biol. Cell. 3, 373-383.

Whitaker, M. and Irvine, R.F., 1984. Inositol 1,4,5-trisphosphate microinjection activates sea urchin eggs. Nature. 312,636-639. 114

Whitaker, M., Swann, K,, and Crossley, I., 1989. What happens during the latent period at fertilization. 157-171.

White, AM.» W@*#on, S.P., and Gahone, A., 1993. Cychc ADP-ribose-induced Ca^ release from n* brain microsomes. FEES Lett. 318,259-263.

Willmott, N., Sethi, J.K., Walseth, T.F., Lee, H.C., White, A.M., and Gahone, A., 1996. Nitric oxide-induced mobilization of intracellular calcium via the cychc ADP-ribose signaling pathway. J. Biol. Chem. 271,3699-3705.

Wing, M.R., Houston, D., Kelley, G.G., Der, C.J., Siderovski, D P., and Harden, T.K., 2001. Activation of phosphohpase C-epsilon by heterotrimeric G protein betagamma- subunits. J. Biol. Chem. 276,48257-48261.

Wu, D., Jiang, H., Katz, A., and Simon, M.I., 1993. Identification of critical regions on phospholipase C-beta 1 required for activation by G-proteins. J. Biol. Chem. 268, 3704-3709.

Yim, D.L., Opresko, L.K., Wiley, H.S., and Nuccitelli, R., 1994. Highly polarized EGF receptor tyrosine kinase activity initiates egg activation in Xenopus. Dev. Biol. 162, 41-55.

Zhou, C., Qian, X.Q., and Roberts, M.F., 1997. Allosteric activation of phosphatidylinositol- specific phosphohpase C: Specific phosphohpid binding anchors the enzyme to the interface. Biochemistry. 36,10089-10097.

Zocchi, E., Franco, L., Guida, L., Benatti, U., Bargellesi, A., Malavasi, F., Lee, H.C., and De Flora, A., 1993. A single protein immunologically identified as CD38 displays NAD* glycohydrolase, ADP-ribosyl cyclase and cychc ADP-ribose hydrolase activities at the outer surface of human erythrocytes. Biochem. Biophys. Res. Commun. 196, 1459-1465.

Zucker, R.S., Steinhardt, R.A., and Winkler, M.M., 1978. Intracellular calcium release and the mechanisms of parthenogenetic activation of the sea urchin egg. Dev. Biol. 65, 285-295.