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The Carboxyl Terminus of the γ-Subunit of Rod cGMP Phosphodiesterase Contains Distinct Sites of Interaction with the Enzyme Catalytic Subunits and the α-Subunit of Transducin
Journal article   Open access   Peer reviewed

The Carboxyl Terminus of the γ-Subunit of Rod cGMP Phosphodiesterase Contains Distinct Sites of Interaction with the Enzyme Catalytic Subunits and the α-Subunit of Transducin

Nikolai P Skiba, Nikolai O Artemyev and Heidi E Hamm
The Journal of biological chemistry, Vol.270(22), pp.13210-13215
06/02/1995
DOI: 10.1074/jbc.270.22.13210
PMID: 7768919
url
https://doi.org/10.1074/jbc.270.22.13210View
Published (Version of record) Open Access

Abstract

The interaction between the GTP-bound form of the transducin α-subunit (Gαt) and the γ-subunit (Pγ) of cGMP phosphodiesterase (PDE) is a key event in effector activation during photon signal transduction. The carboxyl-terminal half of Pγ is involved in interaction with Gαt as well as in inhibition of PDE activity. Here we have utilized a combination of synthetic peptide and mutagenesis approaches to localize specific regions of the carboxyl-terminal region of Pγ interacting with Gαt and Pαβ and have determined residues involved in inhibition of PDE activity. We found that synthetic peptide corresponding to residues 68-87 of Pγ completely inhibit trypsin-activated PDE. The peptide Pγ-63-87 bound to GαtGTPγS with a Kd of 2.5 μM, whereas the binding of Pγ-68-87 to GαtGTPγS was approximately 15-fold less (Kd= 40 μM) suggesting that carboxyl-terminal Pγ region 68-87 contains a site for interaction with Pαβ and also a part of the αt binding site. To map Gαt and Pαβ sites more precisely within the carboxyl-terminal region, a set of carboxyl-terminal mutants was generated by site-directed mutagenesis. Deletion of residues 63-69 and 70-76 diminished the binding of mutants to αt while binding to carboxyl-terminally truncated mutants lacking up to 11 amino acid residues was unchanged. In contrast, carboxyl-terminal truncations of Pγ from Δ1 to Δ11 resulted in a gradual decrease of its inhibitory activity. Thus, the extreme carboxyl-terminal hydrophobic sequence -Ile86-Ile87 together with 9 adjacent residues provides inhibitory interaction of Pγ with Pαβ. The carboxyl-terminal GαtGTPγS binding site of Pγ is different from but adjacent to its PDE inhibitory site. During the visual transduction process, GαtGTP likely binds to this region of Pγ inducing a displacement of the extreme carboxyl terminus from the inhibitory site on Pαβ, leading to PDE activation.

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