Purified Phospholipase C Isozymes by G-Protein {alpha} Subunits Purification and Characterization of Recombinant G16{alpha} From Sf9 Cells: Activation of T Kozasa,

A cDNA encoding Gi6a, the a subunit of a heterotrimeric guani nucleotide-binding protein, was expressed in Sf9 ceUls using recombinant baculovirus. G16a in membrane extracts of Sf9 ceUs activated phospholipase C-.81 (PLC-p81) in the presence of guanosine 5'-[Vthio]triphosphate; the system could not be activated by A13+, Mg2+, and F-. The Gi6a in the cYtosolic fraction of Sf9 ceHs did not stimulate PLC-l1. Concurrent expression of the G-protein Py subunit complex increased the amount of G16a in Sf9 cell membranes. The guanosine 5'-['thioJtriphosphate-activated form of G16a was purifled from cholate extracts of membranes from cells expressing G16., and the G-protein P2 and "Y subunits. G16a activated PLC-p1, PLC-p2, and PLC-83 in a manner essentially i uishable from that of Gqa. Gl,-mediated activation of PLC-p1 and PLC-.83 greatly exceeded that of PLCP2. G16a did not activate PLC-V1 or PLC-81. Thus, two distantly related members of the Gqa family, Gqa and G16a, have the same ability to activate the known isoforms ofPLC-P. The a subunits of heterotrimeric, signal-transducing guanine nucleotide-binding proteins (G proteins) can be classified into four major groups, based on amino acid sequence relationships and some major functional characteristics (1). The so-called Gqa class has four members-Gqa, Gla, G14a, and G15a/Gl6a (2-5). Within this group, Gqa, Glla, and G14a are very similar to one another. Gl5a and Gl6a, which are now thought to be mouse and human homologs, are more distantly related to Gqa (58% and 57% amino acid identity, respectively); they are 85% identical to each other. All members of the Gqa class lack the cysteine residue near the carboxyl terminus that is the site of pertussis toxin-catalyzed ADP-ribosylation of members of the Gi class of a subunits. Thus, Gqa proteins are presumed to function in signaling pathways that are immune to disruption by the toxin. Many hormones, neurotransmitters, and growth factors activate certain isozymes of phospholipase C (PLC) and thus stimulate the hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate to yield two intracellular second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (6). PLCs of the y subtype are in general activated by receptors with intrinsic tyrosine kinase activity, whereas PLCs of the P subtype are regulated by G proteins (7). Mixtures ofGqa and Glla isolated from bovine brain or bovine liver were first shown to activate PLC-f31 upon combination of the purified proteins (8, 9). We have further demonstrated that a mixture of brain Gqa/G1ia and recombinant Gqa (rGqa) and rGii., purified individually after baculovirus-directed expression in Sf9 insect cells, can activate purified PLC-,Bl, PLC-,82, and PLC-/f3, but not PLC-yl or PLC-81 (10, 11). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. A cDNA that encodes Gl6a was cloned from a human promyelocytic leukemia (HL-60) cell library (5). In contrast to Gqa and Gll., which are expressed ubiquitously, Gl6a is synthesized predominantly in hematopoietic cells. We have purified the guanosine 5'-[y-thio]triphosphate (GTP[yS])activated form of G16a after expression in Sf9 cells and characterized its interactions with various isozymes of PLC. MATERIALS AND METHODS Construction of Recombinant Baculovirus and Sf9 Cell Culture. A 1.25-kb DNA fragment containing the complete coding sequence for Gl6a was excised from pKSG16 (5) by digestion with Bal I and Xba I and was subcloned into the Sma I and Xba I sites of the baculovirus transfer vector pVL1393. The resulting plasmid and linearized AcRP-lacZ viral DNA were transfected into Sf9 cells by lipofection. Recombinant virus was plaque-purified as described (12). Positive viral clones were identified by their capacity to direct the expression of G16a protein in Sf9 cells, which was detected by immunoblotting with anti-G16. antiserum B861. Sf9 cells were grown in suspension as described (11). Measurement of PLC Activity. PLC activity was estimated as described (8, 11). In most cases PLC activity is expressed as pmol of InsP3 per min per ng of PLC. To quantify purification of G16a (Table 1), PLC specific activity is expressed as nmol of InsP3 per min per mg of protein sample containing G16a. The amount of PLC used per assay was 2.9 ng for bovine brain PLC-13l, 1 ng for recombinant PLC-.31, 8 ng for recombinant PLC-/2, 0.2 ng for bovine brain PLC-,f3, 2 ng for PLC-yl, and 2 ng for PLC-61. Assays were performed for 5 min with PLC-f81 and for 10 min with the other phospholipases. Purification of G16. from Sf9 Cells. Sf9 cells (8-liter culture; 1.5 x 106 cells per ml) were infected with recombinant baculoviruses (2-5 plaque-forming units per cell) encoding G16a and G-protein (2 and 72 subunits (13). Cells were harvested after 48 hr by centrifugation at 1000 x g for 10 min and were suspended in 800 ml of ice-cold lysis buffer [50mM NaHepes, pH 7.2/1 mM EDTA/3 mM EGTA/5 mM MgCl2/3 mM dithiothreitol/50 mM NaCl with the protease inhibitors [phenylmethanesulfonyl fluoride, 20 pg/ml; 7-amino-l-chloro-3-tosylamido-2-heptanone ("tosyllysine chloromethyl ketone"), 20 pg/ml; L-1-tosylamido-2-phenylethyl chloromethyl ketone, 30 pg/ml; and lima bean trypsin inhibitor, 30 pug/ml]. Cells were lysed by nitrogen cavitation (Parr bomb) at 500 psi (1 psi = 6.89 kPa) for 30 min at 4°C. Cell lysates were centrifuged at 750 x g for 10 min to remove intact cells and nuclei, and the supematant was further Abbreviations: AMF, AP3+/Mg2+/F-; GTP[yS], guanosine 5'-[' thio]triphosphate; InsP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C; r (prefix), recombinant. §To whom reprint requests should be addressed.