AMINO-ACID-RESIDUES IN THE 3RD ALPHA-HELIX OF GROWTH-HORMONE INVOLVEDIN GROWTH-PROMOTING ACTIVITY

The third amphiphilic alpha-helix of GH has been found to be an import ant motif in the biological activities of the molecule. To further cha racterize this growth-promoting domain, three bovine (b) GH analogs we re designed: one contained a scrambled third amphiphilic alpha-helix ( SAH); a second contained a scrambled hydrophilic region of the helix ( SAP); and a third contained a scrambled hydrophobic region of the heli x (SAB). Transgenic mice that expressed these mutated bGH genes were p roduced. SAH transgenic mice displayed a phenotype identical to nontra nsgenic littermates. SAB transgenic mice grew slightly larger than non transgenic littermates but remained smaller than bGH transgenic mice. On the other hand, SAP transgenic mice exhibited a dwarf phenotype. We subsequently generated individual amino acid substitutions in the hyd rophilic region of the helix. The results from the growth rates of cor responding transgenic mice demonstrated that most bGH analogs with ind ividual amino acid substitution within the third alpha-helix retained wild type-like growth-promoting activity except those with alterations at positions 115, 119, 122, and 123. Together these residues are pred icted to form a cleft in the helix. To further substantiate the import ance of the cleft, we deleted Gly 119 (Delta 119). This resulting bGH analog was inactive in vivo as well as in in vitro assays. These resul ts indicated that the primary structure of the third alpha-helix is cr itical for GH's growth-promoting activity and Gly 119 is a crucial ami no acid in this region. Three adjacent amino acids, Asp 115, Ala 122, and Leu 123, also contribute to the growth-enhancing ability of the mo lecule.