Three cytoplasmic loops of rhodopsin interact with transducin.
Rhodopsin is a member of an ancient class of receptors that transduce signals through their interaction with guanine nucleotide-binding proteins (G proteins). We have mapped the sites of interaction of rhodopsin with its G protein, which by analogy suggests how other members of this class of receptors may interact with their G proteins. Three regions of rhodopsin's cytoplasmic surface interact with the rod cell G protein transducin (Gt). These are (i) the second cytoplasmic loop, which connects rhodopsin helices III and IV, (ii) the third cytoplasmic loop, which connects rhodopsin helices V and VI, and (iii) a putative fourth cytoplasmic loop formed by amino acids 310-321, as the carboxyl-terminal sequence emerges from helix VII and anchors to the lipid bilayer via palmitoylcysteines 322 and 323. Evidence for these regions of interaction of rhodopsin and Gt comes from the ability of synthetic peptides comprising these regions to compete with metarhodopsin II for binding to Gt. A spectroscopic assay that measures the "extra MII" caused by Gt binding was used to measure the extent of binding of Gt in the presence of competing peptides. The three peptides corresponding to the second, third, and fourth cytoplasmic loops competed effectively with metarhodopsin II, exhibiting Kd values in the 2 microM range; 11 additional peptides comprising all remaining surface regions of rhodopsin failed to compete even at 200 microM. Any two peptides that were effective competitors showed a synergistic effect, having 15 times higher effectiveness when mixed than when assayed separately. A mathematical model was developed to describe this behavior.
Institut für Biophysik und Strahlenbiologie der Universitat Freiburg, Federal Republic of Germany.
B
B
König
A
A
Arendt
J H
JH
McDowell
M
M
Kahlert
P A
PA
Hargrave
K P
KP
Hofmann
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D003593:Cytoplasm / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D009842:Oligopeptides / Q000138:chemical synthesis; D010786:Photoreceptor Cells / Q000378:metabolism; D011487:Protein Conformation; D012168:Retinal Pigments / Q000378:metabolism*; D012243:Rhodopsin / Q000378:metabolism*; D013053:Spectrophotometry; D015293:Transducin / Q000378:metabolism*
D009842:Oligopeptides; D012168:Retinal Pigments; D012243:Rhodopsin; D015293:Transducin