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 ANALOGS

被引:11
作者
ROWLINSON, SW
BARNARD, R
BASTIRAS, S
ROBINS, AJ
SENN, C
WELLS, JRE
BRINKWORTH, R
WATERS, MJ
机构
[1] UNIV QUEENSLAND,CTR MOLEC BIOL & BIOTECHNOL,BRISBANE,QLD 4072,AUSTRALIA
[2] UNIV ADELAIDE,DEPT BIOCHEM,ADELAIDE,SA 5001,AUSTRALIA
[3] BRESATEC LTD,ADELAIDE,SA 5001,AUSTRALIA
[4] UNIV QUEENSLAND,CTR DRUG DESIGN & DEV,BRISBANE,QLD 4072,AUSTRALIA
关键词
D O I
10.1021/bi00205a008
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Ca-45(2+) 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 (K-d 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.
引用
收藏
页码:11724 / 11733
页数:10
相关论文
共 42 条
[1]   3-DIMENSIONAL STRUCTURE OF A GENETICALLY ENGINEERED VARIANT OF PORCINE GROWTH-HORMONE [J].
ABDELMEGUID, SS ;
SHIEH, HS ;
SMITH, WW ;
DAYRINGER, HE ;
VIOLAND, BN ;
BENTLE, LA .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1987, 84 (18) :6434-6437
[2]   USE OF CALCIUM DEPENDENCE AS A MEANS TO STUDY THE INTERACTION BETWEEN GROWTH-HORMONES AND THEIR BINDING-PROTEINS IN RABBIT LIVER [J].
BARNARD, R ;
WATERS, MJ .
BIOCHEMICAL JOURNAL, 1988, 250 (02) :533-538
[3]   SOLUBLE FORMS OF THE RABBIT ADIPOSE-TISSUE AND LIVER GROWTH-HORMONE RECEPTORS ARE ANTIGENICALLY IDENTICAL, BUT THE INTEGRAL MEMBRANE FORMS DIFFER [J].
BARNARD, R ;
ROWLINSON, SW ;
WATERS, MJ .
BIOCHEMICAL JOURNAL, 1990, 267 (02) :471-477
[4]   MONOCLONAL-ANTIBODIES TO THE RABBIT LIVER GROWTH-HORMONE RECEPTOR - PRODUCTION AND CHARACTERIZATION [J].
BARNARD, R ;
BUNDESEN, PG ;
RYLATT, DB ;
WATERS, MJ .
ENDOCRINOLOGY, 1984, 115 (05) :1805-1813
[5]   A STRATEGY FOR OPTIMIZING CHARGE REVERSAL MUTAGENESIS OF ION-PAIRS IN HORMONE-RECEPTOR OR ENZYME-SUBSTRATE COMPLEXES [J].
BARNARD, R .
PROTEIN ENGINEERING, 1993, 6 (04) :455-459
[6]   AN ELECTROSTATIC MODEL FOR THE INTERACTION BETWEEN GROWTH-HORMONE AND ITS RECEPTOR INVOLVING CHELATION OF CA-2+ TO THE HUMAN GROWTH-HORMONE MOLECULE [J].
BARNARD, R ;
ROWLINSON, SW ;
WATERS, MJ .
JOURNAL OF THEORETICAL BIOLOGY, 1989, 140 (03) :355-367
[7]   A SYSTEMATIC MUTATIONAL ANALYSIS OF HORMONE-BINDING DETERMINANTS IN THE HUMAN GROWTH-HORMONE RECEPTOR [J].
BASS, SH ;
MULKERRIN, MG ;
WELLS, JA .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (10) :4498-4502
[8]   EQUILIBRIUM DENATURATION OF RECOMBINANT PORCINE GROWTH-HORMONE [J].
BASTIRAS, S ;
WALLACE, JC .
BIOCHEMISTRY, 1992, 31 (38) :9304-9309
[9]   HEMATOPOIETIC RECEPTORS AND HELICAL CYTOKINES [J].
BAZAN, JF .
IMMUNOLOGY TODAY, 1990, 11 (10) :350-354
[10]   PORCINE GROWTH-HORMONE RECEPTOR CDNA SEQUENCE [J].
CIOFFI, JA ;
WANG, X ;
KOPCHICK, JJ .
NUCLEIC ACIDS RESEARCH, 1990, 18 (21) :6451-6451