Growth hormone receptor is a target for presenilin-dependent gamma-secretase cleavage.
Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.
Cowan
Jon W
JW
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294-0012, USA.
Wang
Xiangdong
X
Guan
Ran
R
He
Kai
K
Jiang
Jing
J
Baumann
Gerhard
G
Black
Roy A
RA
Wolfe
Michael S
MS
Frank
Stuart J
SJ
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D000111:Acetylcysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D015536:Down-Regulation; D005347:Fibroblasts / Q000378:metabolism; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry; D051379:Mice; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D053764:Presenilin-1; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011986:Receptors, Somatotropin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008565:Membrane Proteins; C508853:PSEN1 protein, human; D053764:Presenilin-1; D061988:Proteasome Inhibitors; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; C067713:lactacystin; D013006:Growth Hormone; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human; C000606463:Adam17 protein, mouse; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine