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Mapping of effector binding sites of transducin alpha-subunit using G alpha t/G alpha i1 chimeras.

Publication ,  Journal Article
Skiba, NP; Bae, H; Hamm, HE
Published in: J Biol Chem
January 5, 1996

The G protein transducin has been an often-used model for biochemical, structural, and mechanistic studies of G protein function. Experimental studies have been limited, however, by the inability to express quantities of mutants in heterologous systems with ease. In this study we have made a series of G alpha t/G alpha i1 chimeras differing at as few as 11 positions from native G alpha t. Ten chimeras are properly folded, contain GDP, can assume an A1F4(-)-induced activated conformation, and interact with beta gamma t and light-activated rhodopsin. They differ dramatically in their affinity for GDP, from Gi-like (initial rates 225 mumol/mol s) to Gt-like (initial rates 4.9 mumol/mol s). We have used these chimeras to define contact sites on G alpha t with the effector enzyme cGMP phosphodiesterase. G alpha t GTP but not G alpha t GDP activates it by removing the phosphodiesterase (PDE) gamma inhibitory subunit. In solution, G alpha t GTP interacts with PDE gamma (Kd 12 nM), while G alpha t GDP binds PDE gamma more weakly (Kd 0.88 microM). The interaction of G alpha i GDP with PDE gamma is undetectable, but G alpha i GDP-A1F4- interacts weakly with PDE gamma (Kd 2.4 microM). Using defined G alpha t/G alpha i chimeras, we have individuated the regions on G alpha t most important for interaction with PDE gamma in the basal and activated states. The G alpha t sequence encompassing alpha helix 3 and the alpha 3/beta 5 loop contributes most binding energy to interaction with PDE gamma. Another composite P gamma interaction site is the conserved switch, through which the GTP-bound G alpha t as well as G alpha i1 interact with P gamma. Competition studies between PDE gamma and truncated regions of PDE gamma provide evidence for the point-to-point interactions between the two proteins. The amino-terminal 1-45 segment containing the central polycationic region binds to G alpha t's alpha 3 helix and alpha 3/beta 5 loop, while the COOH-terminal region of P gamma, 63-87, binds in concert to the conserved switch regions. The first interaction provides specific interaction with both the GDP- and GTP-liganded G alpha t, while the second one is conserved between G alpha t and G alpha i1 and dependent on the activated conformation.

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Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

January 5, 1996

Volume

271

Issue

1

Start / End Page

413 / 424

Location

United States

Related Subject Headings

  • Transducin
  • Sequence Homology, Amino Acid
  • Recombinant Fusion Proteins
  • Phosphoric Diester Hydrolases
  • Phosphodiesterase Inhibitors
  • Molecular Sequence Data
  • GTP-Binding Proteins
  • Biochemistry & Molecular Biology
  • Binding Sites
  • Amino Acid Sequence
 

Citation

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Skiba, N. P., Bae, H., & Hamm, H. E. (1996). Mapping of effector binding sites of transducin alpha-subunit using G alpha t/G alpha i1 chimeras. J Biol Chem, 271(1), 413–424. https://doi.org/10.1074/jbc.271.1.413
Skiba, N. P., H. Bae, and H. E. Hamm. “Mapping of effector binding sites of transducin alpha-subunit using G alpha t/G alpha i1 chimeras.J Biol Chem 271, no. 1 (January 5, 1996): 413–24. https://doi.org/10.1074/jbc.271.1.413.
Skiba NP, Bae H, Hamm HE. Mapping of effector binding sites of transducin alpha-subunit using G alpha t/G alpha i1 chimeras. J Biol Chem. 1996 Jan 5;271(1):413–24.
Skiba, N. P., et al. “Mapping of effector binding sites of transducin alpha-subunit using G alpha t/G alpha i1 chimeras.J Biol Chem, vol. 271, no. 1, Jan. 1996, pp. 413–24. Pubmed, doi:10.1074/jbc.271.1.413.
Skiba NP, Bae H, Hamm HE. Mapping of effector binding sites of transducin alpha-subunit using G alpha t/G alpha i1 chimeras. J Biol Chem. 1996 Jan 5;271(1):413–424.

Published In

J Biol Chem

DOI

ISSN

0021-9258

Publication Date

January 5, 1996

Volume

271

Issue

1

Start / End Page

413 / 424

Location

United States

Related Subject Headings

  • Transducin
  • Sequence Homology, Amino Acid
  • Recombinant Fusion Proteins
  • Phosphoric Diester Hydrolases
  • Phosphodiesterase Inhibitors
  • Molecular Sequence Data
  • GTP-Binding Proteins
  • Biochemistry & Molecular Biology
  • Binding Sites
  • Amino Acid Sequence