Báo cáo khoa học: Binding of cGMP to the transducin-activated cGMP phosphodiesterase, PDE6,

Binding of cGMP to the transducin-activated cGMP

phosphodiesterase, PDE6, initiates a large conformational

change involved in its deactivation

Akio Yamazaki1,2,3, Fumio Hayashi4

, Isao Matsuura5 and Vladimir A. Bondarenko6

1 Kresge Eye Institute, Wayne State University, Detroit, MI, USA

2 Department of Ophthalmology, Wayne State University, Detroit, MI, USA

3 Department of Pharmacology, Wayne State University, Detroit, MI, USA

4 Department of Biology, Kobe University, Japan

5 Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan Town, Taiwan

6 College of Osteopathic Medicine, Touro University, Henderson, NV, USA

Keywords

cGMP binding; cGMP-binding-dependent

protein conformational change; GAF

domains; G-protein-mediated signal

transduction; PDE

Correspondence

V. A. Bondarenko, College of Osteopathic

Medicine, Touro University, Henderson,

NV 89014, USA

Fax: +1 702 777 1799

Tel: +1 702 777 1806

E-mail: [email protected]

(Received 30 January 2011, revised 17

March 2011, accepted 22 March 2011)

doi:10.1111/j.1742-4658.2011.08104.x

Retinal photoreceptor phosphodiesterase (PDE6), a key enzyme for photo￾transduction, consists of a catalytic subunit complex (Pab) and two inhibi￾tory subunits (Pcs). Pab has two noncatalytic cGMP-binding sites. Here,

using bovine PDE preparations, we show the role of these cGMP-binding

sites in PDE regulation. Pabcc and its transducin-activated form, Pabc,

contain two and one cGMP, respectively. Only Pabc shows [3

H]cGMP

binding with a Kd 50 nM and Pc inhibits the [3

H]cGMP binding. Binding

of cGMP to Pabc is suppressed during its formation, implying that cGMP

binding is not involved in Pabcc activation. Once bound to Pabc,

[

3

H]cGMP is not dissociated even in the presence of a 1000-fold excess of

unlabeled cGMP, binding of cGMP changes the apparent Stokes’ radius of

Pabc, and the amount of [3

H]cGMP-bound Pabc trapped by a filter is

spontaneously increased during its incubation. These results suggest that

Pabc slowly changes its conformation after cGMP binding, i.e. after for￾mation of Pabc containing two cGMPs. Binding of Pc greatly shortens the

time to detect the increase in the filter-trapped level of [3

H]cGMP-bound

Pabc, but alters neither the level nor its Stokes’ radius. These results sug￾gest that Pc accelerates the conformational change, but does not add

another change. These observations are consistent with the view that Pabc

changes its conformation during its deactivation and that the binding of

cGMP and Pc is crucial for this change. These observations also imply that

Pabcc changes its conformation during its activation and that release of Pc

and cGMP is essential for this change.

Structured digital abstract

l PDE6 alpha, PDE6 beta and PDE6 gamma physically interact by molecular sieving (View

interaction)

Abbreviations

GAF, a domain derived from cGMP-regulated cyclic nucleotide phosphodiesterases, certain adenylyl cyclases, the bacterial transcription

factor FhlA; GTPcS, guanosine 5¢-O-(3-thiotriphosphate); IBMX, 1-methyl-3-isobutylxanthine; OS, outer segments of retinal photoreceptors;

PDE, cGMP phosphodiesterase; PMSF, phenylmethylsulfonyl fluoride; Pa and Pb, rod PDE catalytic subunits; Pa¢, cone PDE catalytic

subunit; Pab ⁄ Pc, Pab complexes having an unknown number of Pc; Pd, a prenyl-binding protein; Pc, rod PDE inhibitory subunit; Pc¢, cone

PDE inhibitory subunit; T, transducin.

1854 FEBS Journal 278 (2011) 1854–1872 ª 2011 The Authors Journal compilation ª 2011 FEBS