α Helix Content of G Protein α Subunit Is Decreased upon Activation by Receptor Mimetics

Abstract

To elucidate the mechanism whereby liganded receptor molecules enhance nucleotide exchange of GTPbinding regulatory proteins (G proteins), changes in the secondary structure of the recombinant G i1 ␣ subunit (G i1 ␣) upon binding with receptor mimetics, compound 48/80 and mastoparan, were analyzed by circular dichroism spectroscopy. Compound 48/80 enhanced the initial rate of GTP␥S binding to soluble G i1 ␣ 2.6-fold with an EC 50 of 30 g/ml. With the same EC 50 , the mimetic decreased the magnitude of ellipticity, which is ascribed to a reduction in ␣ helix content of the G i1 ␣ by 7%. Likewise, mastoparan also enhanced the rate of GTP␥S binding by 3.0-fold and decreased the magnitude of ellipticity of G i1 ␣ similar to compound 48/80. In corresponding experiments using a K349P-G i1 ␣, a G i1 ␣ counterpart of the unc mutant in G s ␣ in which Pro was substituted for Lys 349 , enhancement of the GTP␥S binding rate by both activators was quite small. In addition, compound 48/80 showed a negligible effect on the circular dichroism spectrum of the mutant. On the other hand, a proteolytic fragment of G i1 ␣ lacking the N-terminal 29 residues was activated and showed decreased ellipticity upon interaction with the compound, as did the wild-type G i1 ␣. Taken together, our results strongly suggest that the activator-induced unwinding of the ␣ helix of the G protein ␣ subunit is mechanically coupled to the enhanced release of bound GDP from the ␣ subunit.The central role played by trimeric GTP-binding regulatory proteins (G proteins) 1 in signal transduction in membranes has received considerable research attention (reviewed in Refs.1-3). Upon ligand binding, a G protein-coupled receptor promotes the release of GDP from inactive trimeric G␣␤␥, which allows binding of cytosolic GTP to the remaining G␣ subunit, thereby resulting in dissociation of trimeric G␣(GTP)␤␥ complex into active G␣⅐GTP and a ␤␥ subunit complex. In this activation process of G protein, the release of bound GDP is of particular interest as it is the rate-limiting step (4). The analyses of x-ray crystallographic structures of the ␣ subunit of G t and G i1 have indicated the presence of two domains, i.e. a GTPase (or Ras-like) domain comprised of ␣ helices and ␤ strands and a highly ␣ helical domain. In addition, the conformational changes induced in the ␣ subunit by nucleotide exchange (GDP 3 GTP␥S) and the mechanism of GTP hydrolysis have been determined (5-9). Conformational changes in the ␣ subunit upon binding with a ␤␥ subunit complex have been determined as well (10, 11). However, the mechanism whereby liganded receptor molecules enhance the GDP release from the ␣ subunit remains unclear, as pointed out previously (3, 12). Likewise, the conformational change of the ␣ subunit upon receptor binding is unknown. The use of physicochemical methods to gain further insight into these key reactions presents difficulties due to the facts that (i) only small amounts of G protein-coupled receptor proteins are expressed in cells, and (ii) no method exists ...

Calnuc, an EF-Hand Ca 2+ binding protein, specifically interacts with the C-terminal α5-helix of Gαi3

“…2 mM of each of four synthetic peptides and in vitro translated [ 35 S]G␣i3 were incubated with calnuc-Sepharose 4B in the presence of 15 mM Mg 2ϩ ͞2 mM Ca 2ϩ as described in Fig. 2 A, (30). Therefore, it seems reasonable to speculate that abolishing G␣i3's binding to calnuc by deletion of its C-terminal 12 amino acids may be caused by disruption of the ␣5 helix.…”

How do basic secretagogues activate mast cells?

“…Follow-up studies corroborated the concept and demonstrated that numerous CADs act in a similar manner as mastoparan (Higashijima et al 1990;Mousli et al 1990b;Hagelüken et al 1995a;Breitweg-Lehmann et al 2002). Elegant mechanistic studies identified the interaction sites of mastoparan on Gproteins and revealed that mastoparan acts as a GPCR mimetic to promote GDP dissociation from G-protein α-subunits (Higashijima et al 1990;Weingarten et al 1990;Higashijima and Ross 1991;Tanaka et al 1998). Overall, the CAD studies with purified G-proteins corroborated the conclusions of studies obtained with intact cells.…”

Crystallization and preliminary X-ray crystallographic analysis of the receptor-uncoupled mutant of Gαi1

“…Between GST and G i1 , a PreScission Protease (GE Healthcare Biosciences) cleavage site (LEVLFQGP) was introduced. The expression plasmid for the K349P mutant of G i1 was prepared by ligating the DraI/SalI fragment of the pET24a(+)/K349P-G i vector (Tanaka et al, 1998), which has Lys349 replaced by Pro in His 10 -G i , into the DraI/SalI sites of pET24a(+)/GST-WT-G i1 , thus producing pET24a(+)/GST-K349P-G i1 . After confirmation of the sequences, the resultant expression plasmids were transformed into Escherichia coli BL21(DE3).…”

“…NP_037277; gene ID 25686), was amplified using the pET24a(+)/ His 10 -G i vector (Tanaka et al, 1998) as the template, with forward primer 5 0 -ATGGGCTGCACACTGAGCGCTGAGGAC-3 0 and reverse primer 5 0 -CCGGTCGACTTAGAAGAGACCACAGTCTT-TTAG-3 0 . The amplified DNA fragment was blunted and digested with SalI.…”

“…Such mutants are known for G s (the unc mutant; Haga et al, 1977;Sullivan et al, 1987) and for G i1 (Tanaka et al, 1998). These proteins have a proline residue in place of the sixth (basic) residue from the C-terminus (Arg342 for G s and Lys349 for G i1 ).…”