Attenuation of GTPase activity of recombinant G(o) alpha by peptides representing sequence permutations of mastoparan.

Abstract

There is convincing evidence that the cyto.plasmnic domains of multisning receptors interact with guanine nucleotide-binding proteins (G proteins). What are the rules governing these interactions? In an attempt to answer this question, we focused our attention on mastoparan, an amphiphilic tetradecapeptide from wasp venom, and on nine of its variants, produced by sequence permutation, which have altered amphiphilicity or no amphiphilici at all. Mastoparan enhances the GTPase activity of recombinant G~a 5-fold in phospholipid vesicles. Like mastoparan, four of the synthetic variants can form amphiphilic a-helices and two of them indeed stimulate the GTPase activity of the G protein, whereas the other two have no effect. This confirms that the activation of certain G proteins by a number of peptides is mainly due to their cationic amphiphilicity. However, this structural feature is clearly not sufficient. The relative orientation ofthe positively charged residues as well as that of the hydrophobic side chains appear to be of fundamental importance. The other five peptides are not amphiphlc and do not enhance the rate of GTP hydrolysis. Surprisingly, three of them almost completely inhibit the G protein's intrinsic GTPase activity. This finding is of interest because of the possible role differential regulation of G protein activity can play in cellular functions.The guanine nucleotide-binding proteins (G proteins) that link cell surface receptors to cytosolic effectors in signal transduction comprise a family of apy heterotrimers associated with the plasma membrane. Upon activation by a liganded receptor the a-subunit binds GTP, dissociates from by, and interacts with one or more effectors (for recent reviews see refs. 1-4). Most known G protein-coupled receptors are characterized by seven hydrophobic transmembrane domains (multispanning receptors), but there is increasing evidence that indicates that certain receptors for growth factors, with a single transmembrane domain, also interact with G proteins (5).The G protein coupling sites ofthe multispanning receptors presumably are located in the second and third cytoplasmic loops (6-8). Synthetic peptides with sequences corresponding to segments of the cytoplasmic loops of the f2-adrenergic receptor have been tested for their effect on adenylate cyclase in erythrocyte membranes. Results suggest that parts of the second, third, and fourth intracellular loops interact with the G protein (9). In contrast, two peptides, comprising the N-and C-terminal 15 amino acids ofthe third intracellular loop of the P2-adrenergic receptor, stimulate the GTPase activity of a recombinant a-subunit of G, ( Because of its amphiphilic nature, MP assumes an a-helical structure when bound to phospholipids, with the positively charged residues exposed to the aqueous medium (13). Presumably, the multispanning receptor regions that interact with G proteins also form cationic amphiphilic a-helices (14).Are there interactions that do not cause stimulation of G protein activity and, ins...

G-protein ligands inhibit in vitro reactions of vacuole inheritance.

“…Mas7[Q4;Qll] is the best available control for excluding membrane-disturbing side effects: Mas17, which is commonly used for this purpose, does not assume an a-helical structure in the presence of membrane and thus binds to them only weakly (Higashijima et al, 1990). Two other Mas derivatives, the Mas-derived peptides 2 and 7 (Oppi et al, 1992), which do not stimulate G-proteins in vitro, were also inactive in the fusion assay.…”

“…8 A). The mastoparan derivatives masX and mas7 (Higashijima et al, 1990) and synthetic mastoparan-derived peptide 8 (Oppi et al, 1992), previously shown to also be potent G-protein activators, inhibited inter-vacuole fusion with approximately the same efficiency as mastoparan. The synthetic mastoparan-derivative masl7 (Higashijima et al, 1990) and the mastoparan-derived peptides 2 and 7 (Oppi et al, 1992; data not shown for peptide 7), all of which do not stimulate G-proteins in vitro, inhibited fusion slightly and then only at high concentrations ( Fig.…”

“…Mastoparan, a wasp venom 14-mer peptide, was a very useful tool in these studies. This peptide stimulates the exchange of GTP for guanosine 5'-diphosphate (GDP) on some or-subunits of heterotrimeric G-proteins by binding to their COOH termini and mimicking an agonist-activated receptor (Higashijima et al, 1988(Higashijima et al, , 1990Mousli et al, 1990;Weingarten et al, 1990;Tomita et al, 1991;Oppi et al, 1992). Thus, binding ofmastoparan constitutively stimulates these heterotrimeric G-proteins, inhibiting in vitro ER-to-Golgi transport (Schwaninger et al, 1992), endocytosis (Carter et al, 1993), endosome fusion (Colombo et al, 1992), exocytosis in chromaltin cells (Vitale et al, 1993), and apical transport in epithelial cells (Pimplikar and Simons, 1993; for a recent review see Bomsel and Mostov, 1992).…”

Differential regulation of G protein α‐subunit GTPase activity by peptides derived from the third cytoplasmic loop of the α2‐adrenergic receptor

“…The expression of recombinant Go~ was verified by SDS/PAGE of induced lysates on 12.5% slab gels according to Laemmli [12] after Coomassie blue staining, as well as after immunoblot analysis using a 1 : 10,000 dilution of a rabbit antiserum specific for Go0~ [13], provided by Dr. Graeme Milligan (University of Glasgow, Scotland). The purification of the recombinant G,,ot from bacterial lysates was carried out as previously reported [14]. The recombinant G~ot subunit (G~c~-s, 45 kDa form), purified according to a slightly modified protocol [15], was a gift from Drs.…”

“…Upon receptorligand interaction, GDP is exchanged for GTP on the G protein's ~-subunit, which subsequently dissociates from the receptor and the fl), dimer and modulates the activity of ion channels or effectors. Following GTP hydrolysis, GceGDP reassociates with fly on the inner surface of the cell membrane [14].…”