Identification of residues outside the two binding sites that are critical for activation of the lactogenic activity of human growth hormone.
Human growth hormone (hGH) binds lactogenic or somatotrophic receptors, creating active heterotrimeric complexes. Comparison of hGH structures, either free or bound to a single lactogenic or somatotrophic receptor, shows binding is associated with structural changes. Changes in hGH structure are unique when binding either lactogenic or somatotrophic receptors and they influence the spatial arrangement of residues constituting the second receptor-binding site. Using site-directed mutagenesis, we identified a contiguous set of largely hydrophobic residues that forms a motif communicating between the two receptor-binding sites of hGH. The residues are external to the receptor-binding epitopes and were identified when their mutation reduced site 2 function without changing site 1 function. The motif includes Phe44, Leu93, Tyr160, Leu163, and Tyr164, located in two hydrophobic clusters between the receptor-binding sites. Their mutation to Glu disrupts hydrophobic interactions and reduces lactogenic activity between 4.7- and 85-fold with little effect on somatotrophic activity or spectroscopic properties. These differential effects indicate that loss of lactogenic activity is not a result of global mis-folding. We propose the loss of lactogenic activity results from disruption of specific hydrophobic clusters that disables the site 1 binding-induced structuring of the second receptor-binding site.
Duda
Karen M
KM
Ohio State Biochemistry Program, Columbus 43221, USA.
Brooks
Charles L
CL
D019943:Amino Acid Substitution; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019382:Human Growth Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010928:Placental Lactogen / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D011994:Recombinant Proteins; D019382:Human Growth Hormone; D010928:Placental Lactogen