Rational design of receptor-specific variants of human growth hormone.

Abstract

Human growth hormone (hGH) binds to both the growth hormone (GH) and the prolactin (PRL) receptors. Competition experiments followed by mutational analysis show that the epitope on hGH for hPRL receptor consists of strong determinants in the middle of helix 1 (comprising residues His-18, His-21, and Phe-25), a loop region (including , and the central portion of helix 4 (containing residues Arg-167, Lys-168, Lys-172, Glu-174, . When these residues are mapped on a structural model of hGH, they form a patch that overlaps but is not identical to that previously determined for the hGH receptor. Three of these side chains (His-18, His-21, and Glu-174) are ligands for binding Zn2+, which is required for high-affinity hGH-hPRL receptor complex formation. By introducing pairs of mutations into hGH that exploit the zinc dependency for hPRL receptor binding and remove side chains in the nonoverlapping regions, we have shifted the binding preference toward the hGH receptor by a factor of 34,000 or toward the hPRL receptor by a factor of 150 without substantial loss in binding affinity for the preferred receptor. The energetic effects of the individual mutations are additive within the double mutants, suggesting that each functions independently and does not introduce gross perturbations in structure. Such receptorselective variants of hGH should be useful molecular probes to link specific receptor binding events to the various biological activities of hGH.Human growth hormone (hGH) elicits a myriad of biological effects including linear growth, lactation, nitrogen retention, diabetogenic and insulin-like effects, and macrophage activation (1-4). Each of these effects begins with the interaction of hGH with specific cellular receptors (5). Thus far, the only cloned human genes whose products bind hGH are the hGH receptor from liver (6) and the human prolactin (hPRL) receptor from mammary gland (7). However, there may be other receptors that bind hGH, including the placental lactogen (PL) receptor (8).The hGH and hPRL receptors (6, 7) contain an extracellular hormone binding domain (consisting of 246 and 211 residues, respectively) that share 32% sequence identity, a single transmembrane domain, and a cytoplasmic domain that differs widely in sequence and length. Nonglycosylated forms of the extracellular binding domains of the hGH and hPRL receptors (hGHbp and hPRLbp, respectively) can be expressed in large quantities in Escherichia coli. These highly purified recombinant binding proteins retain the same affinity for hGH and specificity as their natural glycosylated counterparts (9, 10) and are extremely useful reagents for efficient assessment of the binding affinity for mutants of hGH (9-13).It is not known whether the binding sites on hGH for the hGH and hPRL receptors are identical or which receptor (or combination of receptors) is responsible for which pharmacological effect. To address these issues, we have mapped binding determinants on hGH for the hGH and hPRL receptors and showed that they overlap but are no...

Growth hormone augments superoxide anion secretion of human neutrophils by binding to the prolactin receptor.

“…Among those homologous receptors to which HuGH can bind, only two have been cloned, the HuGH-R (10, 11) and the HuPRL-R (12,13), although there are likely to be others such as the receptor for placental lactogen (14). HuGH residues which are critical for binding to either the HuGH-R or HuPRL-R have been identified by analyzing segment-substituted and single residue-substituted HuGH variants, produced by site-directed mutagenesis techniques, with a competitive binding assay using the extracellular binding proteins derived from the receptors (15)(16)(17)(18). Additionally, the variants were used to demonstrate that zinc mediates binding of HuGH to the HuPRL-R (15).…”

“…Additionally, the variants were used to demonstrate that zinc mediates binding of HuGH to the HuPRL-R (15). Three amino acids ofthe HuGH molecule, residues 18 (His), 21 (His), and 174 (Glu), coordinate with residue 188 (His) of the HuPRL-R to form the four zinc ligands. These zinc ligands appear to be critical for determining the binding preference of HuGH because mutants that alter these positions (15) bind preferentially to somatogenic rather than to lactogenic receptors ( 18).…”

“…binding affinity for the HuGH-or HuPRL-binding protein (BP) as described (17,18). The binding affinities of HuGH variants that were designed to discriminate between the HuGH-R and HuPRL-R are described in Table I. Neutrophil isolation.…”

Improving the pharmacokinetics/pharmacodynamics of prolactin, GH, and their antagonists by fusion to a synthetic albumin-binding peptide

“…The effects of hPRL are mediated via the PRL receptor (PRLR; Goffin et al 1994) and human GH (hGH) via the GH receptor (GHR; Ultsch et al 1994) and PRLR (Cunningham & Wells 1991, Somers et al 1994. Introduction of a single amino acid substitution into GH and PRL results in analogs that behave as competitive hormone antagonists (Chen et al 1990, Fuh et al 1992, Fuh & Wells 1995, Goffin et al 1996, Ramamoorthy et al 2001).…”

Discovery and mechanism of action of pegvisomant

“…The eight substitutions in binding site 1 are H18D, H21N, R167N, K168A, D171S, K172R, E174S, I179T (20,21). It has recently been suggested that the crucial changes are K168A and K172R, both Lys residues.…”