Involvement of a metalloprotease in low-affinity nerve growth factor receptor truncation: inhibition of truncation in vitro and in vivo.
The mechanism of low-affinity NGF receptor (LNGFR) truncation was investigated in cultured Schwann cells. Affinity labeling of Schwann cells with 125I-NGF or metabolic labeling with 35S-cysteine showed that truncated NGF receptor (NGF-Rt) was derived from the cell surface form of the receptor. Addition of full-length, exogenous NGF receptor (M(r) = 80 kDa) to Schwann cell membranes resulted in cleavage of the exogenous substrate to NGF-Rt. Investigations into the mechanism of truncation revealed that metalloprotease inhibitors such as phenanthroline, bathophenanthroline, and 8-hydroxyquinoline (8-OHQ) blocked LNGFR truncation in a concentration-dependent fashion. Inhibitors of other protease classes had no effect on truncation. In addition, truncation did not occur at 4 degrees C. It was found that truncation could also occur in Schwann cell membrane preparations, indicating that the putative protease was membrane bound and closely associated with the LNGFR. Metal reconstitution experiments revealed a strong preference toward zinc for the truncating activity, with iron and manganese having slight reconstitution activity in phenanthroline-quenched membranes. To determine if apparent truncation could be inhibited in vivo, the metalloprotease inhibitor 8-OHQ was administered to neonatal rats. 8-OHQ resulted in decreased urine and blood NGF-Rt levels and increased the sciatic nerve LNGFR content; this effect was dose dependent. In adult rats with sciatic nerve crush lesions, 8-OHQ (30-300 mg/kg, t.i.d.) significantly enhanced the rate of sensory neuron regeneration as assessed by the nerve pinch assay. This was accompanied by increased levels of LNGFR in distal nerve segments. These results suggest that Schwann cells possess a metalloprotease-like activity that serves to cleave LNGFR from the surface of these cells. We propose that the putative metalloprotease represents a novel mechanism by which the Schwann cell regulates this particular cell surface protein. Furthermore, increasing the amount of Schwann cell surface LNGFR appears to be of functional significance in that sensory nerve regeneration can be enhanced by inhibition of truncation.
DiStefano
P S
PS
Neuroscience Research Division, Abbott Laboratories, Abbott Park, Illinois 60064.
Chelsea
D M
DM
Schick
C M
CM
McKelvy
J F
JF
D000345:Affinity Labels; D000818:Animals; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000502:physiology*; D009409:Nerve Crush; D009416:Nerve Regeneration / Q000187:drug effects; D009475:Neurons, Afferent / Q000187:drug effects / Q000502:physiology; D015125:Oxyquinoline / Q000494:pharmacology; D010618:Phenanthrolines / Q000494:pharmacology; D017475:Receptors, Nerve Growth Factor / Q000737:chemistry / Q000502:physiology*; D012583:Schwann Cells / Q000378:metabolism; D012584:Sciatic Nerve / Q000187:drug effects / Q000378:metabolism
D000345:Affinity Labels; D010618:Phenanthrolines; D017475:Receptors, Nerve Growth Factor; C006686:bathophenanthroline; D015125:Oxyquinoline; D008666:Metalloendopeptidases