The Amino Terminus of the Fourth Cytoplasmic Loop of Rhodopsin Modulates Rhodopsin-Transducin Interaction

• Published in: The Journal of biological chemistry 2000-01, Vol.275 (3), p.1930-1936
• Main Authors: Marin, Ethan P., Krishna, A.Gopala, Zvyaga, Tatyana A., Isele, Juergen, Siebert, Friedrich, Sakmar, Thomas P.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; b2-adrenergic receptors; Cattle; Cell Membrane - chemistry; Conserved Sequence; Cytoplasm - metabolism; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Palmitic Acid - metabolism; Peptides - metabolism; Protein Binding; Protein Structure, Tertiary; Rhodopsin - chemistry; Rhodopsin - genetics; Rhodopsin - metabolism; Spectrometry, Fluorescence; Time Factors; Transducin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.3.1930

Rhodopsin is a seven-transmembrane helix receptor that binds and catalytically activates the heterotrimeric G protein transducin (Gt). This interaction involves the cytoplasmic surface of rhodopsin, which comprises four putative loops and the carboxyl-terminal tail. The fourth loop connects the carboxyl end of transmembrane helix 7 with Cys322 and Cys323, which are both modified by membrane-inserted palmitoyl groups. Published data on the roles of the fourth loop in the binding and activation of Gt are contradictory. Here, we attempt to reconcile these conflicts and define a role for the fourth loop in rhodopsin-Gt interactions. Fluorescence experiments demonstrated that a synthetic peptide corresponding to the fourth loop of rhodopsin inhibited the activation of Gt by rhodopsin and interacted directly with the α subunit of Gt. A series of rhodopsin mutants was prepared in which portions of the fourth loop were replaced with analogous sequences from the β2-adrenergic receptor or the m1 muscarinic receptor. Chimeric receptors in which residues 310–312 were replaced could not efficiently activate Gt. The defect in Gtinteraction in the fourth loop mutants was not affected by preventing palmitoylation of Cys322 and Cys323. We suggest that the amino terminus of the fourth loop interacts directly with Gt, particularly with Gαt, and with other regions of the intracellular surface of rhodopsin to support Gt binding.