The surface of visual arrestin that binds to rhodopsin.
OBJECTIVE
The binding of visual arrestin to phosphorylated, activated rhodopsin serves as a model for studying the inactivation process of a large class of G-protein coupled receptor systems. In this study, we combine the use of insertional mutagenesis, fluorescence labeling, and scanning alanine mutagenesis to identify the surface of interaction between arrestin and rhodopsin.


METHODS
The ten amino acid myc tag (EQKLISEEDL) was inserted in eleven loop structures that connect betastrands and the tagged arrestins were heterologously expressed in yeast. Binding competition assays were performed with these proteins, using an anti-myc monoclonal antibody. Site specific cysteines were also substituted in selected loop structures in arrestin. These cysteines were labeled with a fluorescent reporter to assess the proximity of the introduced cysteine with rhodopsin in the bound complex.


RESULTS
Competitive inhibition of arrestin binding to light activated, phosphorylated rhodopsin with an anti-myc antibody showed that all competitive sites lay along a single surface encompassing the N- and C-terminal domains. Fluorescence labeling of these loop structures and subsequent interaction with rhodopsin indicates close apposition of loops 68-78 and 248-253 to rhodopsin in the receptor bound state. Scanning mutagenesis of loop 248-253 implicates Ser-251 and/or Ser-252 as a potential interaction point with rhodopsin.


CONCLUSIONS
Our results clearly suggest a surface of arrestin to which rhodopsin binds upon light activation and phosphorylation. This surface encompasses elements from both the N- and C-terminal domains of arrestin.
Smith
W Clay
WC
Department of Ophthalmology, University of Florida, Gainesville, FL, USA. csmith@eye1.eye.ufl.edu
Dinculescu
Astra
A
Peterson
James J
JJ
McDowell
J Hugh
JH
D019393:Arrestin / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D012243:Rhodopsin / Q000378:metabolism*
D019393:Arrestin; D010446:Peptide Fragments; D012243:Rhodopsin