title: Endoplasmic reticulum-associated degradation of growth hormone receptor in Janus kinase 2-deficient cells.
abstract: A key factor governing cellular sensitivity to GH is cell surface GH receptor (GHR) abundance, which is affected transcriptionally and posttranscriptionally. Mature cell surface GHR abundance is regulated by constitutive and inducible metalloproteolysis and constitutive endosomal/lysosomal degradation. We previously found that Janus kinase 2 (JAK2)-deficient GHR-expressing cells have a greater precursor/mature GHR ratio, exhibit diminished inducible metalloproteolysis, and have a cytoplasmic domain-containing GHR fragment called the basal remnant (by virtue of comigration on SDS-PAGE with the inducible, metalloprotease-generated remnant). Herein we examined the mechanism of generation of basal remnant in JAK2-deficient cells, asking whether it originates from precursor vs. mature receptor and which protease(s) catalyzes its appearance. Prolonged metalloprotease inhibitor treatment or small interfering RNA knockdown of TNF-alpha converting enzyme (TACE) and a disintegrin and metalloprotease-10 (ADAM10) (both implicated in inducible GHR proteolysis) did not reduce basal remnant, indicating its generation is not metalloprotease dependent. However, a mutant GHR resistant to metalloprotease cleavage did not yield basal remnant when expressed in JAK2-deficient cells, suggesting common structural determinants for generation of the inducible remnant and the non-metalloprotease-generated basal remnant seen in JAK2-deficient cells. Treatment of JAK2-deficient cells with a proteasome inhibitor, but not two separate lysosome inhibitors, dramatically decreased basal remnant, accompanied by decreased precursor GHR and increased mature GHR abundance. Disruption of endoplasmic reticulum-to-Golgi transport with brefeldin A (BFA) also reduced basal remnant, and washout of BFA allowed regeneration of basal remnant along with GHR precursor. Notably, BFA washout in the presence of cycloheximide blocked both basal remnant and precursor GHR reappearance, but BFA washout in the presence of lactacystin blocked only basal remnant reappearance, suggesting that basal remnant is generated proteasome dependently from precursor GHR. Collectively, our data suggest that JAK2, by association with GHR in the secretory pathway, blunts proteasome activity-dependent discrete GHR cleavage and endoplasmic reticulum-dependent degradation of the precursor receptor. In so doing, JAK2 enables efficient processing of precursor receptor to mature GHR.
authors.affiliation: University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham, AL 35294-0012, USA.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Loesch
authors.forename: Luqin
authors.initials: L
authors.lastname: Deng
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Wang
authors.forename: Kai
authors.initials: K
authors.lastname: He
authors.forename: Jing
authors.initials: J
authors.lastname: Jiang
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Frank
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000502:physiology; D000072198:ADAM17 Protein; D000643:Ammonium Chloride / Q000494:pharmacology; D045744:Cell Line, Tumor; D019483:Disintegrins / Q000235:genetics / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D053614:Janus Kinase 2 / Q000235:genetics* / Q000502:physiology; D018942:Macrolides / Q000494:pharmacology; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000502:physiology; D045726:Metalloproteases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D034622:RNA Interference; D011986:Receptors, Somatotropin / Q000378:metabolism* / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology
chemical_list: D019483:Disintegrins; D018942:Macrolides; D011986:Receptors, Somatotropin; D000643:Ammonium Chloride; C040929:bafilomycin A1; D013006:Growth Hormone; D053614:Janus Kinase 2; D045726:Metalloproteases; D051722:ADAM Proteins; D020781:Matrix Metalloproteinase 1; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; D046988:Proteasome Endopeptidase Complex