Abstract
Human growth hormone (hGH) stimulates somatogenic and lactogenic actions through the GH and prolactin (PRL) receptors, respectively. In contrast, non-primate GHs stimulate only somatotropic action. Phe44, of the human GH sequence is present in all hormones stimulating lactogenic action and absent in all hormones stimulating only somatotropic action. We speculate that the presence of Phe44 is a feature necessary for specifying lactogenic activity. In this report, the role of Phe44 was investigated by its deletion or substitution with alanine or leucine. Deletion of Phe44 or substitution with leucine did not significantly change the structure of hGH as determined by circular dichroism, absorbance, and fluorescence spectroscopies. In contrast, substitution of alanine perturbed the structure. Deletion of Phe44 reduced binding affinity for the lactogenic receptor, resulting in a reduced activation. Substitution with either alanine or leucine partially restored lactogenic receptor binding affinity, which correlated with the hormones' activity in the Nb2 rat lymphoma cells. All the recombinant hGHs had similar somatotropic activities in FDC-P1 cells transfected with the hGH receptor. These data indicate that the hydrophobic side chain of Phe44 is required for lactogenic receptor binding and activation but is unnecessary for somatotropic action.
Highlights
Growth hormones (GH)1 are members of the GH/prolactin (PRL) family which promote a diverse group of biochemical and physiological processes [1] through the GH receptor [2]
The proteins co-migrated with National Hormone and Pituitary Program (NIH) Human growth hormone (hGH) on SDS polyacrylamide gel electrophoresis under reducing conditions and were observed to be greater than 95% homologous (Fig. 1)
Deletion of Phe44 in methionyl-hGH reduced lactogenic activity and receptor binding affinity but retained somatotropic activity and receptor binding affinity when compared with the wild-type hGH (Table I)
Summary
Growth hormones (GH) are members of the GH/prolactin (PRL) family which promote a diverse group of biochemical and physiological processes [1] through the GH receptor [2]. Alanine scanning of hGH residues that contact either the hGH or hPRL receptor has identified two overlapping sets of residues critical for providing the free energy necessary to facilitate binding These data sets have been used to engineer variants of hPRL [11] and human placental lactogen [14] to bind the hGH receptor at site 1 with affinities close to that of wild-type hGH. Phe is located in the middle of mini-helix 1 and is involved in a hydrophobic interaction with leucine 157 and tyrosines 160 and 164 of the amino terminus of helix 4 This mini-helix is buried at the site 1 receptor interface upon binding to either receptor with a significant 2.5 Å movement toward the amino terminus of helix 4 when hGH is bound to the lactogenic receptor rather than the somatotropic receptor [3]. The positional effects were studied by replacement of Phe with alanine or the more hydrophobic leucine
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