Growth Hormone (GH) and a GH Antagonist Promote GH Receptor Dimerization and Internalization

Abstract

Apocytochrome c is synthesized in the cytoplasm, transported to the mitochondrial intermembrane space, and subsequently covalently attached to heme in a reaction catalyzed by the enzyme cytochrome c heme lyase. We have investigated the amino acid sequences in cytochrome c which are required for mitochondrial import, using a systematic series of site-directed alterations of the CYC7-H3 gene which encodes iso-2-cytochrome c in the yeast Saccharomyces cerevisiae. Import of the altered apocytochromes c was assayed in yeast strains that overexpressed cytochrome c heme lyase. Under these conditions, there was efficient mitochondrial accumulation of forms of apocytochrome c which are incapable of having heme covalently attached. In fact, all apocytochromes c containing deletions located to the carboxyl-terminal side of His27 efficiently accumulated in the mitochondria of strains overexpressing heme lyase, even though all but one of these deletion-containing proteins were incapable of heme attachment. A minimum length of polypeptide chain at the extreme amino terminus of cytochrome c, rather than any specific sequence element in this region, appears to be required for efficient mitochondrial import. Certain amino acid substitutions in the region extending from Gly15 to Leu18, at residue Phe19 and at residue His27, lead to reduced mitochondrial import of apocytochrome c, resulting from stalling of the altered apocytochrome c in partially imported states.

Ligand-independent growth hormone receptor dimerization occurs in the endoplasmic reticulum and is required for ubiquitin system-dependent endocytosis

“…The observed complexes most likely comprise receptor dimers, analogous to the EpoR (21), the epidermal growth factor (EGF) receptor (31), MHC class II ␣ and ␤ molecules (32), and type I and II transforming growth factor ␤ receptors (33). The data are consistent with earlier crosslinking studies with GH or GH antagonist, revealing a large ternary protein complex of one ligand and two receptor molecules (17,18).…”

“…There, GH binding to the complex induces a conformational change, which enables activation of JAK2 molecules. This model is in agreement with the findings that a monovalent GH antagonist can be found in a ternary complex with two GHR molecules but is unable to initiation signal transduction (17,18). The observed inhibition of cellular responses at high GH levels (e.g., the bell-shaped dose-response curve) (41) is most likely the result of the formation of a signaling-incompetent (GH) 2 -(GHR) 2 complex by the simultaneous binding of GH molecules by way of site 1 to the two GHR molecules of the complex.…”

“…More evidence was provided by crosslinking studies with 125 I-labeled GH or GH antagonist. Complexes similar in size and corresponding to a ligand:GHR stoichiometry of 1:2 were detected for both ligands (17,18). Taken together, these data predict the presence of preformed GHR dimers at the cell surface as was also recently proposed for the Epo receptor (EpoR) based on crystallographic data (19,20) and Abmediated immunofluorescence copatching of epitope-tagged EpoRs (21).…”

“…2B, lane 4). Because only ternary complexes composed of one ligand and two GHR molecules have been identified in crosslinking experiments (17,18), we postulate that the observed interaction between wtGHR and mutant GHR molecules is a result of heterodimer formation. The specificity of the immunoprecipitation was determined by transient expression of GHR(1-369; HA-His-6-Myc) into untransfected or GHR (1-434)-expressing ts20 cells.…”

Subcellular Localization and Internalization of the Four Human Leptin Receptor Isoforms

“…HLR-15 possesses a good consensus dileucine motif in its unique cytoplasmic tail, but HLR-67 does not have any obvious endocytic signals. Even these two HLR isoforms were internalized more slowly than other cytokine receptors, including IL-6 receptor hetero-oligomers (23, 47), the growth hormone receptor (48,49), the prolactin receptor (43), and EPO-R (44, 45). However, all of the leptin receptors were internalized more rapidly than the IL-6 receptor in the absence of functional gp130 (47).…”