Tài liệu Báo cáo Y học: The solution structure and activation of visual arrestin studied by

The solution structure and activation of visual arrestin studied

by small-angle X-ray scattering

Brian H. Shilton1

, J. Hugh McDowell2

, W. Clay Smith2 and Paul A. Hargrave2,3

1

Department of Biochemistry, University of Western Ontario, London, Ontario, Canada; 2

Departments of Ophthalmology and 3

Biochemistry and Molecular Biology University of Florida, Gainesville, Florida, USA

Visual arrestin is converted from a
basal state to an

activated state by interaction with the phosphorylated

C-terminus of photoactivated rhodopsin (R*), but the

conformational changes in arrestin that lead to activation

are unknown. Small-angle X-ray scattering (SAXS) was

used to investigate the solution structure of arrestin and

characterize changes attendant upon activation. Wild-type

arrestin forms dimers with a dissociation constant of

60 lM. Small conformational changes, consistent with

local movements of loops or the mobile N- or C-termini

of arrestin, were observed in the presence of a phospho￾peptide correspondingto the C-terminus of rhodopsin,

and with an R175Q mutant. Because both the phospho￾peptide and the R175Q mutation promote bindingto

unphosphorylated R*, we conclude that arrestin is acti￾vated by subtle conformational changes. Most of the

arrestin will be in a dimeric state in vivo. Using the

arrestin structure as a guide [Hirsch, J.A., Schubert, C.,

Gurevich, V.V. & Sigler, P.B. (1999) Cell 97, 257–269], we

have identified a model for the arrestin dimer that is

consistent with our SAXS data. In this model, dimeriza￾tion is mediated by the C-terminal domain of arrestin,

leavingthe N-terminal domains free for interaction with

phosphorylated R*.

Keywords: visual arrestin; rhodopsin; G-protein coupled

receptor signalling; small-angle X-ray scattering; solution

structure.

The first event in the visual cycle is activation of rhodopsin

by light. Photoactivated rhodopsin (R*) initiates a signal

transduction cascade that culminates in membrane hyper￾polarization and the sensation of light (reviewed in [1]). The

sensitivity of the system requires that the signal transmitted

by R* be rapidly attenuated. This is accomplished by a two￾step process involvingphosphorylation of the C-terminus of

R* and bindingby arrestin. Phosphorylation somewhat

decreases the ability of R* to signal transducin. Rapid shut￾off of R* signallingis then accomplished by bindingof

arrestin to photoactivated phosphorylated rhodopsin (R*P

[2–5]).

Arrestin plays a critical role in visual signalling by

completely blockingthe ability of R*P to bind and activate

transducin. Arrestin is present in rod cells at high concen￾trations [6] and therefore a mechanism must exist that

prevents arrestin from inappropriately associatingwith R*.

In fact, arrestin shows very little propensity to bind to R*

until the C-terminal region of R* becomes phosphorylated.

Thus, the C-terminal peptide appears to act as a
switch

that, once phosphorylated, converts arrestin into a state that

is able to bind to R*. The effects of rhodopsin’s phospho￾rylated C-terminal peptide can be mimicked by a synthetic

phosphopeptide or even certain point mutations: both

wild-type arrestin in the presence of the synthetic phospho￾peptide [7], and arrestin-R175Q on its own [8] are able to

bind to unphosphorylated R* and abrogate signalling to

transducin.

The crystal structure of arrestin is known [9,10], but it

is not clear how bindingof the phosphorylated C-terminal

peptide of rhodopsin promotes tight complex formation

between arrestin and R*. One possibility is that bindingof

phosphopeptide leads to a conformational change in

arrestin that increases its affinity for R*. Conformational

changes in arrestin can take place in solution, as

demonstrated by changes in the proteolytic digestion

pattern that result from phosphopeptide binding[7] or by

heparin binding[11], and changes in cysteine reactivity

due to phosphopeptide bindingor the activatingR175Q

mutation [12]. The nature and extent of the conforma￾tional change that leads to activation of arrestin is not

known. The situation is complicated by the fact that

visual arrestin participates in a monomer–dimer equilib￾rium [13,14]. It has been suggested that the arrestin dimer

may function as an inert storage form of the protein,

which can be recruited by dissociation to terminate the

visual signal [14].

To characterize further the mechanism and nature of

arrestin’s activation, we conducted small-angle X-ray scat￾tering(SAXS) studies of arrestin in solution to measure

directly the quaternary structure and conformation of

arrestin, and changes associated with phosphopeptide

bindingor the R175Q mutation. We demonstrate that the

conformation and oligomeric structure of arrestin are not

drastically altered by either phosphopeptide bindingor by

the R175Q mutation. The arrestin dimer will probably be

Correspondence to B. H. Shilton, Department of Biochemistry, The

University of Western Ontario, London ON, N6A 5C1 Canada.

Fax: + 1 519 6613175, Tel: + 1 519 6614124,

E-mail: [email protected]

Abbreviations: R*, photoactivated rhodopsin; R*P, phosphorylated

and photoactivated rhodopsin; Rg, radius of gyration; S, momentum

transfer equal to 2sinh/L; SAXS, small-angle X-ray scattering.

Note: a web site is available at http://www.biochem.uwo.ca

(Received 24 January 2002, revised 19 June 2002,

accepted 25 June 2002)

Eur. J. Biochem. 269, 3801–3809 (2002)
FEBS 2002 doi:10.1046/j.1432-1033.2002.03071.x