Detergent cell extracts were immunoprecipitated with anti-JAK2. exhibited markedly different sensitivity to inducible GHR proteolysis, which correlated directly to their relative levels of mature TACE vs unprocessed TACE precursor and indirectly to their levels of cellular TIMP3. Our results implicate TIMP3 as a modulator of cell surface GHR abundance and the ability of GH to promote cellular signaling; these modulatory effects may be conferred by endogenous TIMP3 expression as well as exogenous TIMP3 exposure. Furthermore, our analysis suggests that TIMP3, in addition to regulating the activity of TACE, may also modulate the maturation of TACE, thereby affecting the abundance of the active form of the enzyme. GH is a 22-kDa polypeptide vertebrate hormone derived largely from the anterior pituitary gland. GH signals powerful somatogenic and metabolic effects and may have a role in cancer cell behavior (1,C4) by interacting with the cell surface GH receptor (GHR), a 620-residue transmembrane glycoprotein member of the cytokine receptor superfamily (5,C7). GH-induced GHR binding causes activation of the GHR-associated tyrosine kinase, Janus kinase (JAK)-2, and downstream signaling pathways, including the signal transducer and activator of transcription (STAT)-5 Abcc4 pathway (8,C10). Cell surface GHR abundance is a key determinant of GH sensitivity that is regulated by cells by several means (11,C13). Among these mechanisms, GHR is targeted for constitutive and inducible metalloprotease-mediated cleavage that alters surface GHR levels and can modulate GH signaling. GHR metalloproteolysis occurs in the proximal extracellular domain stem region and results in Rifamdin loss of the full-length receptor, appearance of a cell-associated cytoplasmic domain-containing GHR fragment (the remnant protein), and shedding of a soluble GHR extracellular domain (the GH binding protein [GHBP]) (14,C16). In cultured cells, GHR proteolysis can be induced by serum, platelet-derived growth factor, or phorbol-12-myristate-12-acetate (PMA) and results in the desensitization to subsequent GH treatment (17, 18). Similarly, treatment of mice with endotoxin also elicits this proteolytic GHR down-regulation and desensitization to hepatic GH action (19). We previously demonstrated that GHR proteolysis is catalyzed Rifamdin by the TNF- converting enzyme (TACE; ADAM17) (17), a disintegrin and metalloprotease (ADAM) family member first described as the enzyme responsible for the generation of the soluble TNF- through the cleavage of its membrane-bound precursor (20, 21). TACE is noteworthy in that it is a transmembrane protein with a zinc-dependent catalytic region residing in its extracellular domain. In addition to TNF- and GHR, TACE cleaves numerous transmembrane substrates, including amyloid precursor protein, Notch1, and epidermal Rifamdin growth factor family ligands, in their extracellular domains (22,C24). Tissue inhibitors of metalloproteases (TIMP) are soluble proteins that regulate metalloprotease activity by noncovalent interaction with the protease (25). TIMP3 is unique among family members because it is a physiologically relevant specific inhibitor of TACE (26, 27), although the mechanism of TIMP3’s inhibition of TACE is incompletely known. Our prior work established that inducible TACE-mediated proteolysis of GHR is a determinant of cellular GH sensitivity and that pretreatment with a hydroxamate-based chemical TACE inhibitor prevented inducible GHR proteolysis and rendered cells resistant to TACE-mediated GH desensitization (18). In the current study, we examine the relationships between cellular TIMP3 expression, GHR metalloproteolysis, and GH sensitivity. Our results implicate TIMP3 as a modulator of cell surface GHR abundance and the ability of GH to promote cellular signaling; this effect on GHR availability may be conferred by endogenous TIMP3 expression, as well as exogenous TIMP3 exposure. Furthermore, our analysis suggests that TIMP3, in addition to regulating the activity of TACE, may also Rifamdin modulate Rifamdin the maturation of TACE, thereby affecting the abundance of the active form of the enzyme. Materials and Methods Materials Routine reagents were purchased from Sigma-Aldrich Corp unless otherwise noted. Fetal bovine serum, gentamicin sulfate, penicillin, and streptomycin were purchased from BioFluids. Recombinant human GH was kindly provided by Eli Lilly & Co. Antibodies Polyclonal anti-phospho-STAT5.