Proc. Natl. Acad. Sci. USA Vol. 96, pp. 7843–7846, July 1999 Biochemistry Selective interaction of the C2 domains of phospholipase C-␤1 and -␤2 with activated G␣q subunits: An alternative function for C2-signaling modules TIELI WANG*, SRINIVAS PENTYALA†,JOHN T. ELLIOTT*, LOUISA DOWAL*, EKTA GUPTA†,MARIO J. REBECCHI*†, AND SUZANNE SCARLATA*‡ *Department of Physiology and Biophysics and †Department of Anesthesiology, State University of New York, Stony Brook, NY 11794-8661 Communicated by William J. Lennarz, State University of New York, Stony Brook, NY, May 4, 1999 (received for review March 16, 1999) ABSTRACT Phospholipase C (PLC)-␤1 and PLC-␤2 are subunits, allowing for interactions with the catalytic do- regulated by the Gq family of heterotrimeric G proteins and main (3). ␣ 2؉ contain C2 domains. These domains are Ca -binding mod- In contrast, the C-terminal extension plays a key role in G q ules that serve as membrane-attachment motifs in a number regulation because its deletion preserves intrinsic activity but ␣ of signal transduction proteins. To determine the role that C2 abolishes activation by G q subunits (4–6). Truncation of the ␤ ␤ domains play in PLC- 1 and PLC- 2 function, we measured C terminus also reduces the affinity of the protein for activated ␣ Ϸ the binding of the isolated C2 domains to membrane bilayers. G q subunits 25-fold, but the residual affinity between the We found, unexpectedly, that these modules do not bind to two proteins is still very strong. PLC-␤s are also GAPs membranes but they associate strongly and specifically to (GTPase-activating proteins), and proteins derived from this activated [guanosine 5؅-[␥-thio]triphosphate (GTP[␥S])- C-terminal region have Ϸ20% of the total GAP activity of the ␣ ␤ bound] G q subunits. The C2 domain of PLC- 1 effectively intact enzyme (7). suppressed the activation of the intact isozyme by In this study, we investigate the role of the C2 domains of ␣ ␥ ␣ G q(GTP[ S]), indicating that the C2-G q interaction may PLC-␤ and PLC-␤ . C2 domains have been identified and ␣ ␥ 1 2 be physiologically relevant. C2 affinity for G q(GTP[ S]) was characterized in many proteins to date (for review see refs. 8 ␣ reduced when G q was deactivated to the GDP-bound state. and 9). In general, C2 domains are Ϸ130 residues in length and ␣ ␤␥ Binding to activated G i1 subunits or to G subunits was not consist of a compact ␤-sandwich of two four-stranded ␤-sheets. ␣ ␥ detected. Also, G q(GTP[ S]) failed to associate with the C2 C2 domains generally are found in proteins that interact with ␦ ␣ domain of PLC- , an isozyme that is not activated by G q. lipid membranes that have been grouped into four major ␤ These results indicate that the C2 domains of PLC- 1 and classes (see ref. 8): those that mediate vesicle transport, modify ␤ ␣ PLC- 2 provide a surface to which G q subunits can dock, lipids, regulate GTPase activity, and phosphorylate proteins. leading to activation of the native protein. Most, but not all, C2 domains bind Ca2ϩ, and many are able to target their host protein to lipid membranes. It is notable Phosphatidylinositol-specific phospholipase Cs (PLCs) are that in some cases, such as synaptotagmins, C2 domains ϩ Ca2 -dependent enzymes that catalyze the hydrolysis of phos- mediate calcium-dependent dimerization, binding to target phatidylinositol 4,5-bisphosphate [PIns(4,5)P2] to generate the proteins and to inositol polyphosphates. ␦ two second messengers diacylglycerol and inositol 1,4,5- The crystal structure of PLC- 1 shows that its C2 domain, trisphosphate (for reviews see refs. 1 and 2). These messengers which binds Ca2ϩ, is integrated with the catalytic core, and so then promote the activation of protein kinase C and the release ϩ simple deletion of this domain would be expected to produce of Ca2 from intracellular stores. There are three known an inactive protein. It was proposed that the function of the C2 ␥ ␦ ␤ families of mammalian PLCs ( , , and ), which differ in their domain of PLC-␦ correctly orients the catalytic site to the lipid ␥ 1 regulation. PLC- s are regulated by tyrosine kinase receptors substrate (10). However, recent biochemical studies of mu- ␦ whereas regulators of the PLC- family are not well defined. tated PLC-␦ indicate that this may not be the case, leaving the ␤ 1 The four members of the PLC- family, which we will focus on function of the C2 domain of PLC-␦ unclear (11). Sequence ␣ 1 here, are activated by the subunits from the G q family of homology maps of the C2 domains of PLC-␤, which we have ␤␥ heterotrimeric G proteins as well as G subunits. studied here, show nonconservative replacements of some of ␤ The mammalian PLC- s are modular proteins containing an the Asp residues required for Ca2ϩ binding. N-terminal pleckstrin homology (PH) domain, four elongation ␤ Here, we have expressed the C2 domains of PLC- 1, PLC- factor (EF) hand motifs, a catalytic X/Y domain, a C2 domain, ␤ ␦ 2, and PLC- 1 and studied their ability to bind various and a long, 400-residue C-terminal extension that is unique to membranes in a Ca2ϩ-dependent manner by using fluores- the PLC-␤ family. Although the roles of the EF hand motifs ␦ cence methods. Although only the C2-PLC- 1 showed mem- and C2 domain are unknown, the functions of the PH domain brane binding, we found, surprisingly, that the C2 domains of and C-terminal region have been investigated. Studies of the ␤ ␤ ␣ ␤ ␤ PLC- 1 and PLC- 2 mediate specific association to G q isolated PH domains of PLC- 1 and PLC- 2 show that they subunits, indicating that these domains may mediate G protein have a membrane-binding affinity on the order of the intact signal transduction. enzyme and help to anchor the protein to the membrane surface, allowing for lateral association to G protein subunits ␤ ␤ Abbreviations: PLC, phosphoinositide-specific phospholipase C; PH, (3). The PH domains PLC- 1 and PLC- 2 also have a strong ␥ Ј ␥ ␤␥ ␤␥ pleckstrin homology; GTP[ S], guanosine 5 -[ -thio]triphosphate; affinity for G and may play a key role in docking the G GST, glutathione S-transferase; POPC, 1-palmitoyl 2-oleoyl phos- phatidylcholine; POPS, 1-palmitoyl 2-oleoyl phosphatidylserine; The publication costs of this article were defrayed in part by page charge POPE, 1-palmitoyl 2-oleoyl phosphatidylethanolamine; PIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; C, 7-methoxycoumarin-3- payment. This article must therefore be hereby marked ‘‘advertisement’’ in carboxylic acid; DAB, 4-[4-(dimethylamino)phenylazo]benzoic acid. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed. e-mail: suzanne@ PNAS is available online at www.pnas.org. physiology.pnb.sunysb.edu or [email protected]. 7843 Downloaded by guest on September 30, 2021 7844 Biochemistry: Wang et al. Proc. Natl. Acad. Sci. USA 96 (1999) MATERIALS AND METHODS the labeled proteins to the nonfluorescent lipid was followed ␤ ␤ by the increase of coumarin fluorescence. Binding of C2- 1 Protein Preparation. Preparation of recombinant PLC- 1, ␤ ␤ ␣ ␤ ␥ ␣ and C2- 2 could not be detected even at lipid concentrations PLC- 2,G q, and G 1 2 expressed in Sf9 cells and G i1 as high as 500 ␮M 1-palmitoyl 2-oleoyl phosphatidylcholine expressed in bacteria have been described (12). The integrity (POPC), POPC/1-palmitoyl 2-oleoyl phosphatidylserine of the protein products was assessed electrophoretically and by ␤ ␣ Ј ␥ (POPS) (1:2), or POPC/PIns(4,5)P2 (2%) (data not shown). PLC- activity and G q[guanosine 5 -[ -thio]triphosphate 2ϩ ␥ Addition of 10 mM free Ca did not influence binding. We (GTP[ S])] activation as reported (13). ␦ 2ϩ did find that C2- 1 bound weakly in a Ca -dependent manner C2 domains were prepared by amplifying the coding se- ␤ ␤ to the anionic membranes, but at affinities that were too weak quences for C2-PLC- 1 (residues 663–802), C2-PLC- 2 (res- ␦ to be determined accurately by these methods (partition idues 688–805), and C2-PLC- 1 (residues 615–756) by PCR coefficient, defined as the ratio of the concentration of free and by inserting them into the corresponding sites in the Ͼ and membrane-bound protein, Kp 1 mM). expression vector pGEX (Pharmacia) fused to glutathione The C2 Domains of PLC-␤1 and PLC-␤2 Bind Specifically S-transferase (GST). Escherichia coli BL-21 (DE3) cells, trans- to G␣q Subunits. Because the C2 domain of the PLC-␤ formed with pGEX-C2s expression vectors, were grown in isozymes lies between the catalytic core and the C-terminal super broth containing ampicillin (50 ␮g/ml). GST-C2 domain ␣ region that mediates activation by G q subunits, we tested the expression was induced with 1 mM isopropyl ␤-D-thiogalac- ␣ idea that the C2 domains may serve to link G q to the topyranoside at 18°C, and the bacteria were harvested after ␤ ␤ activation and catalytic domains of the protein. Association 24 h. C2- 1 and C2- 2 were extracted as GST fusion proteins ␣ between the C2 domains and G q subunits was determined by from the soluble fraction of the bacterial lysates by using ␣ labeling the G q with coumarin (C-) and measuring the glutathione-Sepharose 4B resin according to the manufactur- Ͼ transfer of its excited energy to a nonfluorescent energy- er’s protocol (Pharmacia). Proteins were 95% pure, as transfer acceptor covalently attached to the C2 domains, judged by SDS/PAGE analysis. 4-[4-(dimethylamino)phenylazo]benzoic acid (DAB-). Be- ␦ Because C2-PLC- 1 was found mainly in the insoluble cause energy transfer is highly distance dependent, association fraction of the bacterial extracts, inclusion bodies were solu- of the two proteins is observed by the loss of donor fluores- bilized in 8 M urea and slowly diluted to 4 M urea before ␤ cence.