Abstract
The growth hormone (GH) receptor (GHR) binds GH in its extracellular domain and transduces activating signals via its cytoplasmic domain. Both GH-induced GHR dimerization and JAK2 tyrosine kinase activation are critical in initiation of GH signaling. We previously described a rapid GH-induced disulfide linkage of GHRs in human IM-9 cells. In this study, three GH-induced phenomena (GHR dimerization, GHR disulfide linkage, and enhanced GHR-JAK2 association) were examined biochemically and immunologically. By using the GH antagonist, G120K, and an antibody recognizing a dimerization-sensitive GHR epitope, we demonstrated that GH-induced GHR disulfide linkage reflects GH-induced GHR dimerization. GH, not G120K, promoted both GHR disulfide linkage and enhanced association with JAK2. Measures that diminished GH-dependent JAK2 and GHR tyrosine phosphorylation diminished neither GH-induced GHR disulfide linkage nor GH-enhanced GHR-JAK2 association. By using both transient and stable expression systems, we determined that cysteine 241 (an unpaired extracellular cysteine) was critical for GH-induced GHR disulfide linkage; however, GH-induced GHR dimerization, GHR-JAK2 interaction, and GHR, JAK2, and STAT5 tyrosine phosphorylation still proceeded when this cysteine residue was mutated. We conclude GH-induced GHR disulfide linkage is not required for GHR dimerization, and activation and GH-enhanced GHR-JAK2 association depends more on GHR dimerization than on GHR and/or JAK2 tyrosine phosphorylation.
Highlights
From the ‡Department of Medicine, Division of Endocrinology and Metabolism and the §Department of Cell Biology, University of Alabama at Birmingham, the ¶Veterans Affairs Medical Center, Birmingham, Alabama 35294, and the ʈEdison Biotechnology Institute, Ohio University, Athens, Ohio 45701
Extracts were subjected to immunoprecipitation with anti-GHRcyt-AL37 (a rabbit polyclonal serum directed at the growth hormone receptor (GHR) cytoplasmic domain (18)), nonimmune rabbit serum, anti-GHRcyt-mAb, or anti-GHRext
Eluates of these precipitates as well as nonprecipitated detergent extracts were resolved under reducing conditions by SDS-PAGE, and GHRs present in each sample were immunoblotted with anti-GHRcyt (an independently derived rabbit polyclonal serum directed at the receptor cytoplasmic domain (11))
Summary
Vol 274, No 46, Issue of November 12, pp. 33072–33084, 1999 Printed in U.S.A. Disulfide Linkage of Growth Hormone (GH) Receptors (GHR) Reflects GH-induced GHR Dimerization. The growth hormone (GH) receptor (GHR) binds GH in its extracellular domain and transduces activating signals via its cytoplasmic domain Both GH-induced GHR dimerization and JAK2 tyrosine kinase activation are critical in initiation of GH signaling. GHR-JAK2 interaction can occur without GH occupancy of the GHR and can be detected in vitro without tyrosine phosphorylation of JAK2 or the GHR (15); GH treatment of cells augments the degree and/or strength of association of JAK2 with the GHR (7, 15, 18).2 Whereas this enhanced GHR-JAK2 association likely relates to GH-induced JAK2 signaling, the structural basis for this augmentation has yet to be intensively investigated. In this communication, we biochemically and immunologically examine in several GHR-expressing cell lines the relationships between GH-induced GHR dimerization, GHR disulfide linkage, and GHR-JAK2 interaction. By identifying an unpaired extracellular domain cysteine (residue 241) as critical to GH-induced GHR disulfide linkage, we differentiate noncovalent from disulfide-linked GHR dimerization, allowing us to compare these processes as they apply to the GHR with findings for other cytokine and growth factor receptors
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
