Role of the Cytokine-induced SH2 Domain-containing Protein CIS in Growth Hormone Receptor Internalization

Abstract

The cytokine-inducible SH2 domain-containing protein CIS inhibits signaling from the growth hormone (GH) receptor (GHR) to STAT5b by a proteasome-dependent mechanism. Here, we used the GH-responsive rat liver cell line CWSV-1 to investigate the role of CIS and the proteasome in GH-induced GHR internalization. Cell-surface GHR localization and internalization were monitored in GH-stimulated cells by confocal immunofluorescence microscopy using an antibody directed against the GHR extracellular domain. In GH naïve cells, GHR was detected in small, randomly distributed granules on the cell surface and in the cytoplasm, with accumulation in the perinuclear area. GH treatment induced a rapid (within 5 min) internalization of GH⅐GHR complexes, which coincided with the onset of GHR tyrosine phosphorylation and the appearance in the cytosol of distinct granular structures containing internalized GH. GHR signaling to STAT5b continued for ϳ30 -40 min, however, indicating that GHR signaling and deactivation of the GH⅐GHR complex both proceed from an intracellular compartment. The internalization of GH and GHR was inhibited by CIS-R107K, a dominant-negative SH2 domain mutant of CIS, and by the proteasome inhibitors MG132 and epoxomicin, which prolong GHR signaling to STAT5b. GH pulse-chase studies established that the internalized GH⅐GHR complexes did not recycle back to the cell surface in significant amounts under these conditions. Given the established specificity of CIS-R107K for blocking the GHR signaling inhibitory actions of CIS, but not those of other SOCS/CIS family members, these findings implicate CIS and the proteasome in the control of GHR internalization following receptor activation and suggest that CIS-dependent receptor internalization is a prerequisite for efficient termination of GHR signaling. Growth hormone (GH)2 is an important regulator of somatic growth and cellular metabolism. GH exerts its action via the GH receptor (GHR), a transmembrane protein of the cytokine receptor superfamily that activates multiple intracellular signaling pathways, including the STAT, mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase C pathways (1, 2). Upon binding of ligand, the dimeric GHR undergoes a conformational change (3, 4) and induces tyrosine phosphorylation, resulting in activation of the tyrosine kinase JAK2, which catalyzes tyrosine phosphorylation of GHR and of GHR-or JAK2-associated STAT1, STAT3, STAT5a, and STAT5b (5, 6). The tyrosine-phosphorylated STAT proteins dimerize and translocate to the nucleus, where they bind to specific DNA response elements upstream of GH target genes and activate gene transcription (7,8).Many physiological responses to GH are dependent on the temporal pattern of plasma GH stimulation, which is sex-dependent and subject to neuroendocrine control. In the rat model, the adult male plasma GH pattern is characterized by regular pulses of hormone every ϳ3.5-4 h, separated by well defined GH-free intervals, whereas a more continuous plasma GH profile i...

Regulation of Constitutive Mouse Hepatic Cytochromes P450 and Growth Hormone Signaling Components by 3-Methylcholanthrene

“…6). CIS, a specific member of the suppressor of cytokine signaling (SOCS) family, is induced by GH via a STAT5-dependent transcriptional mechanism and plays a role in the negative feedback loop that inhibits GHR signaling (Landsman and Waxman, 2005). MUP2 is a member of the family of ␣2-microglobulin-related liver secretory proteins that form a significant component of protein in mouse urine; the male predominance of MUP2 is also due to pulsatile GH signaling via a STAT5-dependent mechanism (Udy et al, 1997).…”

Crystal structure of the SOCS2–elongin C–elongin B complex defines a prototypical SOCS box ubiquitin ligase

“…6, which is published as supporting information on the PNAS web site). The closely related CIS1 is also associated with the ubiquitination and internalization of GHR, and this activity is lost upon mutation of its SH2 domain (R107K) or in the presence of proteasome inhibitors (28). The involvement of SOCS3 in GHR regulation remains unclear; its mouse knockout is embryonic lethal (29).…”

Aryl hydrocarbon receptor-dependence of dioxin’s effects on constitutive mouse hepatic cytochromes P450 and growth hormone signaling components

“…CIS is induced by GH via a STAT5b-dependent transcriptional mechanism and plays a role in negative feedback inhibition of GH signaling (Landsman and Waxman 2005). MUP2 belongs to the family of α2-microglobulin-related liver secretory proteins and is a significant protein component of mouse urine; pulsatile GH signaling via a STAT5b-dependent mechanism confers malepredominant expression of MUP2 (Udy et al 1997).…”