Growth hormone (GH) insensitivity syndrome due to a GH receptor truncated after Box1, resulting in isolated failure of STAT 5 signal transduction.

Congenital GH insensitivity syndrome (GHIS) is usually the result of a mutation in the extracellular domain of the GH receptor (GHR). We report one of only a small number of mutations so far identified within the intracellular domain of the GHR. The probands are a 53-yr-old woman, height 114 cm (SD score, -8.7), peak GH 45 microg/liter during hypoglycemia, IGF-I 8.0 microg/liter [normal range (N) N 54-389], IGF binding protein-3 16 nmol/liter (N 61-254), GHBP 6.8% (N > 10); and her 57-yr-old brother, height 140 cm (SD score, -6), IGF-I 38.8 micro g/liter (N 54-290), IGF binding protein-3 30 nmol/liter (N 61-196). Both patients were homozygous for a 22-bp deletion in the DNA encoding the cytoplasmic domain of the GHR, resulting in a frameshift and premature stop codon. The resultant GHR is truncated at amino acid 449 (GHR1-449) after Box1, the Janus kinase 2 binding domain of the receptor. Functional studies in HEK293 and Chinese hamster ovary cells show GHR1-449 to have a cellular distribution similar to that of the wild-type GHR, judged by binding of iodinated GH, FACS analysis, and immunocytochemistry. Western blot analysis showed GH-induced phosphorylation of Janus kinase 2, signal transducer and activator of transcription (Stat)3, and Erk2 for both GHR1-449 and wild-type GHR. However, no Stat5 activity was detected in cells expressing GHR1-449, consistent with the fact that GHR1-449 contains no Stat5 binding site. In conclusion, we report two adult siblings with GHIS due to a mutation in the intracellular domain of GHR resulting in a selective loss of Stat5 signaling. Results are consistent with the hypothesis that the loss of signaling through the Stat5 pathway results in GHIS.

cAMP-specific phosphodiesterase HSPDE4D3 mutants which mimic activation and changes in rolipram inhibition triggered by protein kinase A phosphorylation of Ser-54: generation of a molecular model.

Ser-13 and Ser-54 were shown to provide the sole sites for the protein kinase A (PKA)-mediated phosphorylation of the human cAMP-specific phosphodiesterase isoform HSPDE4D3. The ability of PKA to phosphorylate and activate HSPDE4D3 was mimicked by replacing Ser-54 with either of the negatively charged amino acids, aspartate or glutamate, within the consensus motif of RRES54. The PDE4 selective inhibitor rolipram ¿4-[3-(cyclopentoxy)-4-methoxyphenyl]-2-pyrrolidone¿ inhibited both PKA-phosphorylated HSPDE4D3 and the Ser-54-->Asp mutant, with an IC50 value that was approximately 8-fold lower than that seen for the non-PKA-phosphorylated enzyme. Lower IC50 values for inhibition by rolipram were seen for a wide range of non-activated residue 54 mutants, except for those which had side-chains able to serve as hydrogen-bond donors, namely the Ser-54-->Thr, Ser-54-->Tyr and Ser-54-->Cys mutants. The Glu-53-->Ala mutant exhibited an activity comparable with that of the PKA phosphorylated native enzyme and the Ser-54-->Asp mutant but, in contrast to the native enzyme, was insensitive to activation by PKA, despite being more rapidly phosphorylated by this protein kinase. The activated Glu-53-->Ala mutant exhibited a sensitivity to inhibition by rolipram which was unchanged from that of the native enzyme. The double mutant, Arg-51-->Ala/Arg-52-->Ala, showed no change in either enzyme activity or rolipram inhibition from the native enzyme and was incapable of providing a substrate for PKA phosphorylation at Ser-54. No difference in inhibition by dipyridamole was seen for the native enzyme and the Ser-54-->Asp and Ser-54-->Ala mutants. A model is proposed which envisages that phosphorylation by PKA triggers at least two distinct conformational changes in HSPDE4D3; one of these gives rise to enzyme activation and another enhances sensitivity to inhibition by rolipram. Activation of HSPDE4D3 by PKA-mediated phosphorylation is suggested to involve disruption of an ion-pair interaction involving the negatively charged Glu-53. The increase in susceptibility to inhibition by rolipram upon PKA-mediated phosphorylation is suggested to involve the disruption of a hydrogen-bond involving the side-chain hydroxy group of Ser-54.