Use of protein knobs to characterize the position of conserved alpha-subunit regions in lutropin receptor complexes.
Efforts to identify the manner in which human choriogonadotropin (hCG) contacts lutropin receptors (LHR) have been stymied by the complex structure of the hormone and the likelihood that it contacts the receptor at multiple sites. During studies of hCG assembly in mammalian cells, we found that addition of a cysteine to the long disordered beta-subunit COOH terminus (betaCT) enabled it to become cross-linked by a disulfide to cysteines that are substituted for residues in loop alpha2 or in the alpha-subunit COOH terminus (alphaCT). This created a "knob" on the alpha-subunit at the location of the cysteine. Knobs of various sizes and charges were useful for probing surfaces of the alpha-subunit thought previously to contact the LHR. Attachment of the betaCT to residues in loop alpha2 facing loops beta1 and beta3 reduced hormone activity only a few fold revealing that this surface does not participate in essential high affinity receptor contacts, a finding inconsistent with our earlier view of the hCG-LHR complex. In contrast, this approach showed that the opposite surface of loop alpha2 appeared to be nearer the receptor interface. Although attachment of knobs to portions of the alphaCT reduced hormone activity substantially, this finding was difficult to interpret. As discussed, this procedure should be adapted readily to other proteins and may facilitate the introduction of fluorophores, enzymes, or other reagents at specific sites on protein surfaces. It may also permit one to cross-link proteins or to obscure specific protein surfaces during the development of "Trojan Horse" therapeutics.
Department of OB-GYN, Robert Wood Johnson (Rutgers) Medical School, Piscataway, New Jersey 08854, USA.
Yongna
Y
Xing
Win
W
Lin
Mei
M
Jiang
Donghui
D
Cao
Rebecca V
RV
Myers
Michael P
MP
Bernard
William R
WR
Moyle
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D007986:Luteinizing Hormone / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011974:Receptors, LH / Q000737:chemistry*; D015398:Signal Transduction; D014162:Transfection
D004220:Disulfides; D010455:Peptides; D011974:Receptors, LH; D007986:Luteinizing Hormone; D003545:Cysteine