Interaction of Bovine Growth Hormone with Buffalo Adipose Tissue and Identification of Signaling Molecules in Its Action

Results on localization of growth hormone receptor (GHR), interaction of growth hormone (GH) with receptor in buffalo adipose tissue and identification of activated signaling molecules in the action of GH are presented. Bovine GH (bGH) was labeled with fluorescein or biotin. Fluorescein-labelled bGH was used for localization of GHRs in buffalo adipocytes. The receptors were present on the cell surface. The affinity of binding of GH to its receptor was determined by designing an experiment in which buffalo adipose tissue explants, biotinylated GH and streptavidin-peroxidase conjugate were employed. The affinity constant was calculated to be $2{\times}10^8M^{-1}$. The receptor density on adipose tissue was found to be 1 femto mole per mg of tissue. Signalling molecules generated in the action of GH were tentatively identified by employing Western blot and enhanced chemiluminescence techniques using anti-phosphotyrosine antibody. Based on molecular weights of proteins reactive to anti-phosphotyrosine antibody, three signaling molecules viz. insulin receptor substrate, Janus activated kinase (Jak) and mitogen activated protein were tentatively identified. These signaling molecules appeared in a time (incubation time of explants with growth hormone) dependent way. The activation of Jak2 was confirmed by employing anti-Jak2 antibody in a Western blot. The activation of Jak2 occurred during 5 min incubation of buffalo adipose tissue explants with GH and incubation for an additional period, viz. 30 min. or 60 min., resulted in a drastic reduction in activation. The results suggest that Jak2 activation is an early event in the action of GH in buffalo adipose tissue.