Metalloprotease-mediated GH receptor proteolysis and GHBP shedding. Determination of extracellular domain stem region cleavage site.
Growth hormone-binding protein (GHBP) is complexed to a substantial fraction of circulating GH. In humans, rabbits, and other species, GHBP derives from proteolytic shedding of the GH receptor (GHR) extracellular domain. In cell culture studies, stimuli such as phorbol ester, platelet-derived growth factor, or serum induce GHR proteolysis, which concomitantly yields shed GHBP in cell supernatants and a cell-associated cytoplasmic domain-containing GHR remnant. This process is sensitive to metalloprotease inhibition, and genetic reconstitution studies identify tumor necrosis factor-alpha converting enzyme (TACE/ADAM-17), a transmembrane metalloprotease, as a GHR sheddase. Stimuli that induce GHR proteolysis render cells less responsive to GH, but the mechanism(s) of this desensitization is not yet understood. In this study, we mapped the rabbit (rb) GHR cleavage site. We adenovirally expressed a C-terminal epitope-tagged rbGHR lacking most of its cytoplasmic domain, purified the remnant protein induced by the phorbol ester, PMA, and derived the cleavage site by N-terminal sequencing of the purified remnant. The N-terminal sequence, (239)FTCEEDFR(246), matched perfectly the rbGHR and suggests that cleavage occurs eight residues from the membrane in the proximal extracellular domain stem region. Deletion and alanine substitution mutagenesis indicated that, similar to other TACE substrates, the spacing of residues in this region, more than their identity, influences GHR cleavage susceptibility. Further, we determined that PMA pretreatment desensitized a cleavage-sensitive GHR mutant, but not a cleavage-insensitive mutant, to GH-induced JAK2 activation. These results suggest that inducible GHR proteolysis can regulate GH signaling.
Wang
Xiangdong
X
Department of Medicine, University of Alabama at Birmingham, Alabama 35294, USA.
He
Kai
K
Gerhart
Mary
M
Huang
Yao
Y
Jiang
Jing
J
Paxton
Raymond J
RJ
Yang
Shaohua
S
Lu
Chunxia
C
Menon
Ram K
RK
Black
Roy A
RA
Baumann
Gerhard
G
Frank
Stuart J
SJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D016895:Culture Media, Serum-Free; D005110:Extracellular Space / Q000502:physiology; D006801:Humans; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids; D011986:Receptors, Somatotropin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D002352:Carrier Proteins; D016895:Culture Media, Serum-Free; D010446:Peptide Fragments; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; D013755:Tetradecanoylphorbol Acetate; C051815:somatotropin-binding protein