scholarly journals The N Terminus of GTPγS-activated Transducin α-Subunit Interacts with the C Terminus of the cGMP Phosphodiesterase γ-Subunit

2006 ◽  
Vol 281 (10) ◽  
pp. 6194-6202 ◽  
Author(s):  
Jennifer E. Grant ◽  
Lian-Wang Guo ◽  
Martha M. Vestling ◽  
Kirill A. Martemyanov ◽  
Vadim Y. Arshavsky ◽  
...  
Keyword(s):  
Α Subunit ◽  
Cgmp Phosphodiesterase ◽  
Γ Subunit ◽  
C Terminus ◽  
N Terminus

scholarly journals Identification of Heterotrimeric G Protein γ3 Subunit in Rice Plasma Membrane

2018 ◽  
Vol 19 (11) ◽  
pp. 3591 ◽  
Author(s):  
Aki Nishiyama ◽  
Sakura Matsuta ◽  
Genki Chaya ◽  
Takafumi Itoh ◽  
Kotaro Miura ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Higher Plants ◽  
Rice Genome ◽  
Amino Acid Residues ◽  
Subunit Gene ◽  
Α Subunit ◽  
Γ Subunit ◽  
Domain Antibody ◽  
C Terminus ◽  
Liquid Chromatography Tandem Mass

Heterotrimeric G proteins are important molecules for regulating plant architecture and transmitting external signals to intracellular target proteins in higher plants and mammals. The rice genome contains one canonical α subunit gene (RGA1), four extra-large GTP-binding protein genes (XLGs), one canonical β subunit gene (RGB1), and five γ subunit genes (tentatively named RGG1, RGG2, RGG3/GS3/Mi/OsGGC1, RGG4/DEP1/DN1/OsGGC3, and RGG5/OsGGC2). RGG1 encodes the canonical γ subunit; RGG2 encodes the plant-specific type of γ subunit with additional amino acid residues at the N-terminus; and the remaining three γ subunit genes encode the atypical γ subunits with cysteine abundance at the C-terminus. We aimed to identify the RGG3/GS3/Mi/OsGGC1 gene product, Gγ3, in rice tissues using the anti-Gγ3 domain antibody. We also analyzed the truncated protein, Gγ3∆Cys, in the RGG3/GS3/Mi/OsGGC1 mutant, Mi, using the anti-Gγ3 domain antibody. Based on nano-liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the immunoprecipitated Gγ3 candidates were confirmed to be Gγ3. Similar to α (Gα) and β subunits (Gβ), Gγ3 was enriched in the plasma membrane fraction, and accumulated in the flower tissues. As RGG3/GS3/Mi/OsGGC1 mutants show the characteristic phenotype in flowers and consequently in seeds, the tissues that accumulated Gγ3 corresponded to the abnormal tissues observed in RGG3/GS3/Mi/OsGGC1 mutants.

scholarly journals Stability and biological activities of heterodimeric and single-chain equine LH/chorionic gonadotropin variants

2008 ◽  
Vol 40 (4) ◽  
pp. 185-198 ◽  
Author(s):  
Sébastien Legardinier ◽  
Jean-Claude Poirier ◽  
Danièle Klett ◽  
Yves Combarnous ◽  
Claire Cahoreau
Keyword(s):  
Thermal Stability ◽  
Chorionic Gonadotropin ◽  
Sf9 Cells ◽  
Β Subunit ◽  
Single Chain ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Covalently Linked

Recombinant equine LH/chorionic gonadotropin (eLH/CG) was expressed in the baculovirus–Sf9 insect cell system either as a single-chain with the C-terminus of the β-subunit fused to the N-terminus of the α-subunit or as non-covalently linked heterodimers with or without a polyhistidine tag at various locations. All these non-covalently linked eLH/CG variants were secreted as stable heterodimers in the medium of infected Sf9 cells. To assess the influence of the presence and the position of polyhistidine tag on LH bioactivity, we expressed four non-covalently linked tagged heterodimeric eLH/CG variants that were secreted in threefold higher quantities than the single chain. Among them, only two exhibited full in vitro LH bioactivity, relative to untagged heterodimers, namely the one His-tagged at the N-terminus of α-subunit and the other at the C-terminus of the β-subunit both of which are amenable to nickel-affinity purification. Furthermore, single-chain eLH/CG was found to be N- and O-glycosylated but nevertheless less active in in vitro LH bioassays than natural eCG and heterodimeric recombinant eLH/CG. The thermal stability of natural and recombinant hormones was assessed by the initial rates of dissociation from 20 to 90 °C. Heterodimeric eLH/CG from Sf9 cells was found to be as stable as pituitary eLH and serum eCG (T1/2, 74–77 °C). Although Sf9 cells only elaborated short immature-type carbohydrate side chains on glycoproteins, recombinant eLH/CG produced in these cells exhibited stabilities similar to that of pituitary eLH. In conclusion, recombinant heterodimeric eLH/CG exhibits the same thermal stability as natural pituitary LH and its advantages over the single-chain eLH/CG include higher secretion, higher in vitro bioactivity, and reduced potential risk of immunogenicity.

scholarly journals Escherichia coli l-aspartate-α-decarboxylase: preprotein processing and observation of reaction intermediates by electrospray mass spectrometry

1997 ◽  
Vol 323 (3) ◽  
pp. 661-669 ◽  
Author(s):  
Manoj K. RAMJEE ◽  
Ulrich GENSCHEL ◽  
Chris ABELL ◽  
Alison G. SMITH
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Electrospray Mass Spectrometry ◽  
Elevated Temperatures ◽  
Gene Encoding ◽  
Α Subunit ◽  
C Terminus ◽  
N Terminus ◽  
Terminal Amino ◽  
Α Subunits

The Escherichia coli panD gene, encoding l-aspartate-α-decarboxylase, was cloned by PCR, and shown to complement apanD mutant defective in β-alanine biosynthesis. Aspartate decarboxylase is a pyruvoyl-dependent enzyme, and is synthesized initially as an inactive proenzyme (the π-protein), which is proteolytically cleaved at a specific X–Ser bond to produce a β-subunit with XOH at its C-terminus and an α-subunit with a pyruvoyl group at its N-terminus, derived from the serine. The recombinant enzyme, as purified, is a tetramer, and comprises principally the unprocessed π-subunit (of 13.8 kDa), with a small proportion of the α- and β-subunits (11 kDa and 2.8 kDa respectively). Incubation of the purified enzyme at elevated temperatures for several hours results in further processing. Using fluorescein thiosemicarbazide, the completely processed enzyme was shown to contain three pyruvoyl groups per tetrameric enzyme. The presence of unchanged serine at the N-terminus of some of the α-subunits was confirmed by electrospray mass spectrometry (ESMS) and N-terminal amino acid sequencing. A novel HPLC assay for aspartate decarboxylase was established and used to determine the Km and kcat for l-aspartate as 151±16 μM and 0.57 s-1 respectively. ESMS was also used to observe substrate and product adducts trapped on the pyruvoyl group by sodium cyanoborohydride treatment.

scholarly journals Interaction sites of the C-terminal region of the cGMP phosphodiesterase inhibitory subunit with the GDP-bound transducin α-subunit

1999 ◽  
Vol 337 (2) ◽  
pp. 281-288 ◽  
Author(s):  
Yu LIU ◽  
Vadim Y. ARSHAVSKY ◽  
Arnold E. RUOHO
Keyword(s):  
Bound States ◽  
Present Report ◽  
Active Form ◽  
Terminal Region ◽  
Functional Difference ◽  
Α Subunit ◽  
Cgmp Phosphodiesterase ◽  
Interaction Sites ◽  
Γ Subunit ◽  
Inactive Form

In the present report, the region of interaction between the GDP-bound α-subunit of transducin (αt.GTP) and the cGMP phosphodiesterase inhibitory γ-subunit (Pγ) has been studied. It is widely accepted that the αt.GTP is the active form of transducin and that the GDP-bound transducin α-subunit (αt.GDP) is the inactive form. We have reported previously that the binding region of the C-terminal of Pγ on αt.GTP is in a region between the exposed face of the α3 and α4 helices of αt.GTP [Liu, Arshavsky and Ruoho (1996) J. Biol. Chem. 271, 26900–26907]. We now report that N-[(3-[125I]iodo-4-azidophenylpropionamido-S-(2-thiopyridyl)]cysteine ([125I]ACTP)-derivatized Pγ (at Cys-68) reversibly undergoes a unique disulphide exchange of the radioiodinated moiety N-(3-[125I]iodo-4-azidophenylpropionamido)cysteine ([125I]APC) from Cys-68 of Pγ to αt.GDP but not to the guanosine 5´-(γ-thio)-triphosphate (GTP[S])-bound transducin α-subunit (αt-GTP[S]). The specificity of the interaction was demonstrated by the fact that exchange was protected by the functionally active Cys-68 → Ala Pγ mutant, and by pretreatment of the αt.GDP with the βγ-subunit of transducin. Chemical cleavage and amino acid sequencing demonstrated that the [125I]ACTP-derived Pγ specifically transferred the [125I]APC group to Cys-250 and Cys-210 of αt.GDP. These data indicate that the C-terminal region (especially Cys-68–Trp-70) of Pγ interacts with αt.GDP on the exposed interface between α2/β4 and α3/β5 of the α-subunit of transducin. Disulphide exchange was also observed with the α-subunit of holotransducin but this was only approx. 60% of that of pure αt.GDP. The variation in the binding pattern between αt.GDP and αt.GTP with the C-terminal region of Pγ may contribute to the functional difference between the GDP- and GTP-bound states.

scholarly journals Structural basis for distinctive recognition of fibrinogen γC peptide by the platelet integrin αIIbβ3

2008 ◽  
Vol 182 (4) ◽  
pp. 791-800 ◽  
Author(s):  
Timothy A. Springer ◽  
Jianghai Zhu ◽  
Tsan Xiao
Keyword(s):  
Metal Ion ◽  
Structural Data ◽  
Structural Basis ◽  
Side Chain ◽  
Α Subunit ◽  
Integrin Αiibβ3 ◽  
Rgd Motif ◽  
Γ Subunit ◽  
Fibrinogen Binding ◽  
C Terminus

Hemostasis and thrombosis (blood clotting) involve fibrinogen binding to integrin αIIbβ3 on platelets, resulting in platelet aggregation. αvβ3 binds fibrinogen via an Arg-Asp-Gly (RGD) motif in fibrinogen's α subunit. αIIbβ3 also binds to fibrinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the γ subunit (γC peptide). These distinct modes of fibrinogen binding enable αIIbβ3 and αvβ3 to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin αIIbβ3–γC peptide interface, and, for comparison, integrin αIIbβ3 bound to a lamprey γC primordial RGD motif. Compared with RGD, the GAKQAGDV motif in γC adopts a different backbone configuration and binds over a more extended region. The integrin metal ion–dependent adhesion site (MIDAS) Mg2+ ion binds the γC Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca2+ ion binds the γC C terminus, revealing a contribution for ADMIDAS in ligand binding. Structural data from this natively disordered γC peptide enhances our understanding of the involvement of γC peptide and integrin αIIbβ3 in hemostasis and thrombosis.

scholarly journals Residues within the Polycationic Region of cGMP Phosphodiesterase γ Subunit Crucial for the Interaction with Transducin α Subunit

1997 ◽  
Vol 272 (25) ◽  
pp. 15856-15864 ◽  
Author(s):  
Vladimir A. Bondarenko ◽  
Mit Desai ◽  
Salil Dua ◽  
Matsuyo Yamazaki ◽  
Rajesh Haresh Amin ◽  
...  
Keyword(s):  
Α Subunit ◽  
Cgmp Phosphodiesterase ◽  
Γ Subunit
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