Evidence for involvement of the carboxy terminus of helix 1 of growth hormone in receptor binding: use of charge reversal mutagenesis to account for calcium dependence of binding and for design of higher affinity analogues.
In this study we have demonstrated that the C-terminus of helix 1 of porcine GH (pGH) is a receptor-interactive region, thus extending the current binding site model of GH. This was achieved by introducing charge reversal mutations into this region of pGH, which influenced receptor affinity and Ca2+ dependence of binding. The first mutant (R34E pGH, conversion of Arg 34 to Glu) introduced a putative Ca2+ binding site which is present in human GH (hGH) [Barnard et al. (1989) J. Theor. Biol. 140, 355-367] and sits opposite E220 of receptor subunit 1. This mutant exhibited increased Ca2+ dependence of receptor binding but even at optimal Ca2+ did not display higher than wild-type affinity. Introduction of a second Ca2+ binding site adjacent to the first by a second charge reversal (K30E R34E pGH) further increased Ca2+ dependence of binding and also increased affinity for the rabbit GH receptor (2.4 +/- 0.4)-fold relative to wild-type pGH at optimal Ca2+. Equilibrium dialysis and Scatchard analysis of binding of 45Ca2+ to pGH and K30E R34E pGH revealed two Ca2+ binding sites on wild-type pGH and an additional two Ca2+ binding sites on the K30E R34E pGH mutant (Kd 0.5-0.8 mM), as predicted. A third partial charge reversal mutant in the fourth helix (H170D) also led to enhanced Ca2+ dependence of binding, supporting our proposal that E34 and D170 are responsible for the Ca2+ dependence of hGH binding to the rabbit GH receptor. Examination of the crystal structure shows that E34 and D170 are in close proximity and would interact repulsively with a cluster of acidic residues on the receptor consisting of E126, E127, and E220 unless neutralized by Ca2+ or an introduced basic residue. Accordingly, charge reversal at the adjacent pGH residue E33 (E33K pGH) led to a Ca2+ independent (3.0 +/- 0.4)-fold increase in affinity of binding. As well as extending the binding site model of GH, these studies provide a mechanistic explanation for the unique Ca2+ dependence of hGH binding to the rabbit GH receptor. They also indicate that charge reversal can be used to design higher affinity GH analogues and could assist in the mapping of interactive regions in ligand-receptor complexes generally.
Rowlinson
S W
SW
Department of Physiology and Pharmacology, University of Queensland, Brisbane, Australia.
Barnard
R
R
Bastiras
S
S
Robins
A J
AJ
Senn
C
C
Wells
J R
JR
Brinkworth
R
R
Waters
M J
MJ
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D013006:Growth Hormone / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D017433:Protein Structure, Secondary; D011817:Rabbits; D011986:Receptors, Somatotropin / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013552:Swine; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology
D011986:Receptors, Somatotropin; D013006:Growth Hormone; D015032:Zinc; D002118:Calcium