Structure determination of the fourth cytoplasmic loop and carboxyl terminal domain of bovine rhodopsin.

PURPOSE

High resolution structural information is lacking for any member of the class of G-protein receptors. This dearth of structural information extends to virtually all integral membrane proteins. As part of an alternative approach to examining integral membrane protein structure, we are determining the structures of the extramembraneous domains of the G-protein receptor, rhodopsin.

METHODS

The carboxyl terminal domain of bovine rhodopsin was synthesized, containing the last 43 amino acids of the protein sequence (rhoIVe). This sequence included the entire putative fourth cytoplasmic loop as well as a significant portion of helix seven, the transmembrane helix of this receptor to which the carboxyl terminal is attached. The solution structure of rhoIVe was determined by multidimensional 1H nuclear magnetic resonance.

RESULTS

The structure contained a portion of alpha-helix corresponding to the top of transmembrane helix seven of the receptor. This allowed unambiguous docking of the carboxyl terminal domain to a model of the transmembrane domain. Helix seven is longer than suggested by hydropathy analysis. The structure also revealed the fourth cytoplasmic loop. The palmitoylation sites of rhodopsin are located near the deduced membrane surface. However, palmitoylation is not required for formation of this loop.

CONCLUSIONS

The carboxyl terminal of rhodopsin forms a structural domain whose structure can be determined separately from the rest of the protein. This structure reveals the fourth cytoplasmic loop that had been suggested to exist based on the presence of palmitoylation sites in the carboxyl terminal domain. Determination of the structure of all of the cytoplasmic domains of rhodopsin in a manner that allows docking to the structure of the transmembrane domain should permit construction of the entire surface of rhodopsin that interacts with the G-protein, transducin. Additionally, the rhodopsin phosphorylation sites and mutations associated with certain autosomal dominant forms of retinitis pigmentosa can now be located in the three dimensional structure of the carboxyl terminal domain.