Stimulation of phospholipase C‐β₂ by recombinant guanine‐nucleotide‐binding protein βγ dimers produced in a baculovirus/insect cell expression system

Abstract

Recombinant wild-type plyi dimers of signal-transducing guanine nucleotide-binding proteins (G proteins) and plyl dimers carrying a mutation known to block y-subunit isoprenylation (p1ylC71S) were expressed in baculovirus-infected insect cells. Both wild-type and mutant p,yl dimers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic-radiolabeling studies revealed that the soluble plyl preparation contained approximately equal amounts of non-isoprenylated and isoprenylated plyl dimers. Soluble wild-type and mutant ply, dimers and native plyl dimers purified from bovine retina were reconstituted with recombinant phospholipase C-P,. Only isoprenylated plyl dimers were capable of stimulating phospholipase C-p,. The results show that y-subunit isoprenylation and/or additional post-translational processing of the protein are required for py subunit stimulation of phospholipase C.Signal-transducing guanine-nucleotide-binding proteins (G proteins) couple a large variety of receptors to secondmessenger-generating effectors such as adenylyl cyclase, ion channels or phospholipase C (PLC) [l, 21. The activated receptor interacts with the heterotrimeric (a . by) G protein and catalyzes the exchange of GTP for GDP bound to its a subunit. The GTP-bound form of the G protein is likely to dissociate into a GTP-bound a subunit and a free py dimer. In certain cases, e.g. stimulation of various types of adenylyl cyclase or activation of the retinal cGMP phosphodiesterase, the a subunit alone is capable of effector regulation (see [37 for a recent review). In other cases, however, free py dimers may also be involved in controlling effector activity [4, 51. G-protein a, / 3 and y subunits are members of rapidly growing gene families [2]. To date, at least 20 distinct a subunits, four p subunits, and six y subunits are known to exist in mammalian cells [2]. The y subunits of heterotrimeric G proteins belong to a family of proteins and peptides which are post-translationally modified at their carboxy termini by isoprenylation, proteolytic cleavage and methyl esterification (reviewed in [19-221). This family includes a large number of low-molecular-mass GTP-binding proteins, nuclear lamins, fungal mating factors, rhodopsin kinase, and the large subunits of retinal cGMP phosphodiesterase. G-protein y subunits are first either farnesylated (y,) or geranylgeranylated (other y subunits) at a cysteine at position -4 from the carboxy terminus. Following S-isoprenylation, a membrane-bound protease(s) cleaves the three terminal amino acids [23, 241 and the resultant terminal carboxy group is methyl esterified by a membraneous methyltransferase [25].

Liver prenylated methylated protein methyl esterase is the same enzyme as sus scrofa carboxylesterase

“…The dynamic equilibrium may be critical to the biochemical activity of the proteins since the interaction of the prenylated proteins with other macromolecules may be significantly affected by the methylation state of the adjacent −COOH group. This is perhaps more substantial based on the fact that the isoprenyl group of proteins such as the G-protein γ subunits are essential for their coupling of receptor-derived signals to such effector enzymes as the adenylyl cyclases and phospholipase Cβ2 [2,4,5]. Furthermore, an isoprenyl-binding site has been identified using solution NMR on Rho dissociation inhibitor [6].…”

Activation of Signaling Pathways in HL60 Cells and Human Neutrophils by Farnesylthiosalicylate

“…Farnesylcysteine analogs mimic the carboxy-termini of signal-transducing proteins ( y subunits of heterotrimeric G proteins or low-molecular-mass G proteins) and may thus elicit cellular responses by perturbation of interactions of these proteins with specific docking sites on diverse cellular components (IniguezLluhi et al, 1992;Dietrich et al, 1994;Kisselev et al, 1994;van Biesen et al, 1995). Cellular receptors that may be involved in specific interactions with prenylated termini include G-protein-coupled receptors (Kisselev et al, 1994;Sheer and Gierschik, 1995), PLCP (Dietrich et al, 1994) adenylate cyclase (Iniguez-Lluhi et al, 1992) n subunits of G proteins (IniguezLluhi et al, 1992;Higgins and Casey, 1994) and rhodopsin kinase (Premont et al, 1995).…”

“…Cellular receptors that may be involved in specific interactions with prenylated termini include G-protein-coupled receptors (Kisselev et al, 1994;Sheer and Gierschik, 1995), PLCP (Dietrich et al, 1994) adenylate cyclase (Iniguez-Lluhi et al, 1992) n subunits of G proteins (IniguezLluhi et al, 1992;Higgins and Casey, 1994) and rhodopsin kinase (Premont et al, 1995). Blockade or simulation of these interactions by prenylcysteine analogs might in principle account for inhibition or activation of signals transmitted by the prenyl-receptor proteins.…”

“…Farnesylthiosalicylate affected the cellular distribution of Ras by preventing its membrane anchorage, suggesting competition of the analogue with Ras for Ras-docking sites in the membrane . Participation of prenylated termini of signal-transduction proteins in protein-protein interactions has been proposed by others (Marshall, 1993;Iniguez-Lluhi et al, 1992;Dietrich et al, 1994;Kisselev, 1994;Scheer and Gierschik, 1995).…”

Purification and Characterization of Gβγ-responsive Phosphoinositide 3-Kinases from Pig Platelet Cytosol

“…The use of a mutant cDNA encoding a ␥ subunit lacking the essential cysteine residue required for isoprenylation resulted in a shift of the ␤␥ complex to the cytosol and prevented the increase in cellular inositol phosphates. Furthermore, the purification of ␤␥ dimers from baculovirus transfected insect cells has shown that only C-terminally modified ␥ subunits confer PLC␤2-activating function on ␤␥ complexes (35), suggesting that the isoprenylation and carboxyl methylation of ␥ subunits may be important for both membrane location and functionality of the complex. However, the extent of membrane insertion of G␤␥ is unlikely to explain the marked differences between lipid substrates in terms of the cooperativity observed and the extent of activation of PI 3-kinase.…”