Transduction pathways of GH in ovine mammary acini involving regulated and functional growth hormone receptors.

We have investigated the localization and regulation of growth hormone (GH) receptor-related proteins in the ovine mammary gland. Using a new rabbit polyclonal antibody (7122A) directed against the recombinant extracellular domain of GH receptor (GHR-ECD) for western blot assays, we found two bands with apparent molecular weights of 70,000 and 50-60,000 Da in ovine mammary gland solubilized proteins. The 70,000-protein was consistent with a membrane GH receptor form deprived of post-translational modifications such as phosphorylation, glycosylation or ubiquitin binding. The 50-60,000 Da was consistent with soluble GH binding protein, generated by the cleavage of membrane GH receptor. The intensity of related GHR proteins increased slightly throughout mammary gland development and was correlated with the amount of GHR immunoreactivity observed in the mammary gland sections. Moreover, a temporal and spatial regulation of GHR immunoreactivity was found in alveolar epithelial cells. Clearly, marked GHR immunoreactivity was associated with the apical membranes of alveolar epithelial cells at lactation. The up-regulation of related GHR proteins during the differentiation of mammary tissue supports the hypothesis that GH may act specifically via its own receptors. In ovine mammary cells, GH was able to promote a time-dependent activation of MAP kinases such as prolactin (Prl) and placental lactogen (PL). GH was also able to promote slight and transient Stat5 DNA-binding activity. Differences in the time dependence of Stat5 DNA-binding activation by the three different ligands, GH, Prl and PL, were found. All these results emphasize the direct action of GH on ovine mammary cells and highlight the specificity of action of this ligand.