Dimerization of the Extracellular Domain of the Human Growth Hormone Receptor by a Single Hormone Molecule

Abstract

Human growth hormone (hGH) forms a 1:2 complex with the extracellular domain of its receptor-binding protein (hGHbp) as studied by crystallization, size exclusion chromatography, calorimetry, and a previously undescribed fluorescence quenching assay. These and other experiments with protein engineered variants of hGH have led to the identification of the binding determinants for two distinct but adjacent sites on hGH for the hGHbp, and the data indicated that there are two overlapping binding sites on the hGHbp for hGH. Furthermore, the binding of hGH to the hGHbp occurred sequentially; a first hGHbp molecule bound to site 1 on hGH and then a second hGHbp bound to site 2. Hormone-induced receptor dimerization is proposed to be relevant to the signal transduction mechanism for the hGH receptor and other related cytokine receptors.

Characterization of the receptor binding determinants of granulocyte colony stimulating factor

“…Various mechanisms are employed for growth factor signaling upon interaction of each ligand with its receptor. These mechanisms include the 2: 1 receptorligand homodimer seen in the EPO and hGH systems (Cunningham et al, 1991;DeVos et al, 1992;Philo et al, 1996), the 2 1 receptor-ligand heterodimer complex seen for many cytokines including GM-CSF (Altman & Kastelein, 1995), and the 2:2 homodimer complex of interferon gamma with its receptor (Walter et al, 1995;Greenlund et al, 1996). We mutated surface-exposed residues on G-CSF to alanine in order to determine which regions of the four-helix bundle were important for receptor binding and activation.…”

“…Among these residues are all the surface exposed residues on helix D. The lack of important residues on helix D is a marked departure from several other four-helix bundle cytokines. EPO, hGH, IL-2, and IL-4 have residues on the fourth helical bundle that are critically involved in receptor binding (Cunningham et al, 1991;Mott & Campbell, 1995;Matthews et al, 1996). It is also notable that the K40A mutation has no effect on G-CSF activity in binding and proliferation assays.…”

“…Interferon gamma signals as a noncovalent homodimer by recruiting two interferon gamma receptor molecules into a 2:2 receptor-ligand complex (Greenlund et al, 1993;Walter et al, 1995). hGH signaling involves initial binding of hGH to one receptor molecule followed by recruitment of a second receptor to form a 2:l receptor-ligand homodimer (Cunningham et al, 1991;DeVos et al, 1992). Recent evidence suggests that EPO mediates proliferative effects through a similar sequential dimerization process to hGH (Matthews et al ., 1996;Philo et al, 1996).…”

Short Stature Caused by a Mutant Growth Hormone with an Antagonistic Effect.

“…The affinity of the mutant GH for GHBP was significantly higher than that of wild-type GH, and the displacement curve for mutant GH of [125J] human GH binding to human GH receptor had a strange shape, with a shoulder around 10-11 to 10-9 M. The binding of GH to GH receptor is believed to proceed sequentially in the different portion of the GH molecule, first in site 1 and then in site 2 [20]. Our findings suggest that the properties of the mutant GH differ from those of wild-type GH with respect to these affinities in site 1 or site 2.…”

“…This codon is located in the second a helix of the GH molecule behind the binding site 1 to GH receptor [20,21]. The substituted cysteine probably forms new disulfide bonds, changes the charge of the GH molecule, and contributes to a conformational change, causing reduced bioactivity of the mutant GH.…”