Potent peptide analogues of a G protein receptor-binding region obtained with a combinatorial library.
The C terminus of the G protein alpha subunit represents an important site of interaction between heterotrimeric G proteins and their cognate receptors. We have screened a combinatorial peptide library based on the C terminus of the alpha subunit of Gt (340-350) and have identified unique sequences that bind rhodopsin with high affinity. Six of these sequences, as both fusion proteins and synthetic peptides, were significantly more potent than the parent sequence in binding to and stabilization of metarhodopsin II. These sequences provide information about which residues are required for appropriate receptor interaction. We observed that in all the high affinity sequences, a positively charged residue at position 341 was changed to a neutral one. Thus, it appears that the receptor-G protein interaction was designed to be low affinity to ensure efficient catalysis of G protein activation. We also observed Cys-347 and Gly-348 to be invariant, and hydrophobic residues were always located at positions 340, 344, 349, and 350, demonstrating the critical nature of these residues. A composite of the structures of the high affinity sequences was modeled based upon the structure of rhodopsin-bound trNOESY NMR of this region of Gt alpha (Dratz, E. D., Fursteneau, J. E., Lambert, C. G., Thireault, D. L., Rarick, H., Schepers, T., Pakhlevaniants, S., and Hamm, H. E. (1993) Nature 363, 276-280) and provides insight into the complementary G protein-binding surface of the receptor.
Martin
E L
EL
Affymax Research Institute, Palo Alto, California 94304, USA.
Rens-Domiano
S
S
Schatz
P J
PJ
Hamm
H E
HE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D012243:Rhodopsin / Q000031:analogs & derivatives / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D010455:Peptides; D011956:Receptors, Cell Surface; C013110:metarhodopsins; D012243:Rhodopsin; D019204:GTP-Binding Proteins