Endocytosis and lysosomal targeting of epidermal growth factor receptors are mediated by distinct sequences independent of the tyrosine kinase domain.

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) leads to accelerated receptor degradation. Two models have been proposed to explain this. In the first model, induced internalization expands the intracellular pool of receptors, leading to enhanced lysosomal targeting. The second model proposes that activation of intrinsic receptor kinase activity induces inward vesiculation of endosomes, thus interrupting receptor recycling. To test these models, we created EGFR mutants that lack the conserved tyrosine kinase domain, but retain different parts of the distal carboxyl terminus regulatory region. Mutants lacking all distal regulatory sequences underwent slow internalization (0.02 min-1) and turnover (t1/2 approximately 24 h), similar to unoccupied, holo-EGFR. Mutant receptors that lacked the kinase domain, but retained the entire distal regulatory domain, were constitutively internalized and targeted to lysosomes, even in the absence of EGF. The turnover of these receptors (t1/2 approximately 11 h) was similar to that of occupied, kinase-active holo-EGFR (t1/2 approximately 9.5 h). These results show that receptor tyrosine kinase activity is not required for the targeting of EGFR to lysosomes. Receptor mutants which expressed previously identified endocytic sequences underwent rapid internalization. Unexpectedly, enhanced turnover of EGFR mutants required additional sequences located between residues 945 and 991 in the holo-EGFR. Thus, internalization and lysosomal targeting of EGFR are separate processes mediated by distinct sequences. Our results indicate that induced internalization is necessary, but not sufficient, for enhanced EGFR degradation. Instead, down-regulation requires exposure of previously cryptic internalization and lysosomal targeting sequences. Occupied EGFR thus appear to be handled by the endocytic machinery in the same fashion as other constitutively internalized or lysosomally targeted receptors.

Studies on engineered autocrine systems: requirements for ligand release from cells producing an artificial growth factor.

Transplantation of artificial ligand-secreting cells is envisioned as a promising technology in tissue engineering. To achieve practical control of these systems, sufficiently high levels of ligand must be produced to provide adequate receptor binding and signal generation, but ligand spread into adjacent tissue regions must also be controlled. Mathematical models predict that the relative amount of ligand found either in the extracellular medium or bound to cell receptors is governed by ligand and receptor synthesis rates, receptor turnover rate, cell density, and exogenous blocking antibody concentration. To experimentally elucidate the relative contribution of these parameters, we constructed an artificial autocrine system in which all major parameters were under direct experimental control. A synthetic gene for a secretory form of human epidermal growth factor (EGF) was constructed consisting of the mature protein product fused to a foreign signal seqeunce. This was inserted into a vector behind an inducible promoter. Cells lacking endogenous receptors for EGF (EGFR) were first transfected with the gene for the human EGFR and then with the gene for EGF. Cells were selected that expressed high levels of both receptor and ligand. A tetracycline-sensitive promoter system for the artificial EGF gene allowed us to experimentally vary EGF secretion rates 20- to 200-fold. Using this system, we found that no significant amount of EGF was found in the culture medium unless the production rate of the ligand exceeded that of the receptor. Alternately, the use of EGFR-blocking antibodies allowed ligand escape into the medium. Even in the presence of high concentration of antagonistic antireceptor antibodies, however, cells were still able to consume EGF produced in an autocrine fashion. Induction of high levels of EGF production resulted in an almost 90% reduction in total receptor mass through down-regulation, but cells continued to rapidly bind, internalize, and degrade EGF. Our data suggest that cells have an unexpectedly high capacity to both bind and utilize growth factors produced in an autocrine fashion. In addition, interrupting autocrine loops may be more difficult than originally envisioned. Our artificial autocrine system should prove useful in understanding both how these systems are normally regulated and how they can be manipulated for purposes of tissue engineering.

Comparative metabolism and requirement of vitamin K in chicks and rats.

The metabolic basis for the high vitamin K requirement of chicks compared with rats was investigated. When chicks and rats were fed the same diet, containing 500 micrograms phylloquinone/kg, the total amounts of phylloquinone and its epoxide metabolite found in the liver and plasma were similar in both species. However, phylloquinone 2,3-epoxide was present in high concentrations in chick liver and serum but not in rat liver and serum. This metabolite of the vitamin is normally reduced by a hepatic vitamin K epoxide reductase. The activity of this enzyme in chicks was approximately 10% of that in rats, and the inability of chicks to effectively recycle the epoxide of vitamin K seems to be the major factor in its high requirement. Other species differences in vitamin K metabolism were observed. Much higher concentrations of bacterial menaquinones were present in rat feces compared with chick feces, but neither species had appreciable hepatic concentrations of menaquinones. Chicks, but not rats, were found to have a liver concentration of menaquinone-4 that exceeded that of phylloquinone. This vitamer was present even when its recognized precursor, menadione, was not present in the diet, and the data indicate that chicks convert phylloquinone to menaquinone-4 under the conditions of these experiments. The mechanism of this conversion was not established.

Comparative metabolism of phylloquinone and menaquinone-9 in rat liver.

The hepatic turnover of phylloquinone and menaquinone-9 (MK-9) and their relative efficacy in satisfying the dietary requirement for vitamin K were compared in male rats. Rats fed 1.1 mumol phylloquinone/kg diet had higher initial liver and serum vitamin K concentrations than rats fed an equimolar amount of MK-9. The initial rate of hepatic turnover of phylloquinone was two to three times as rapid as that of MK-9. After about 48 h of vitamin K restriction there were no significant differences in hepatic vitamin K concentration of rats fed phylloquinone or MK-9. Phylloquinone was much more effective than MK-9 in maintaining normal vitamin K status at low dietary concentrations (0.2 mumol/kg diet), whereas at high dietary concentrations (5.6 mumol/kg diet) they were equally effective.

Vitamin K status of free-living subjects consuming olestra.

The potential for 20 g olestra/d to affect vitamin K status was assessed in a 6-wk study involving 202 free-living subjects. Functional prothrombin [Simplastin (S)-Ecarin (E) assay] concentrations and classical clotting times were unaffected by olestra. Initial S:E values were 0.80 and 0.79 for the olestra and placebo groups, respectively, compared with a value of 0.92 for normal reference plasma. At week 6 the value was 0.81 for both groups. Mean phylloquinone serum concentrations, expressed as differences from baseline, were not significantly different between groups. Weekly food diaries indicated that the average phylloquinone intake of the subjects was low, approximately 60 micrograms/d. Sensitive measures of vitamin K status were unaffected in a population where any significant decrease in phylloquinone bioavailability should have been reflected in those measures, indicating that 20 g olestra/d in the diet did not affect vitamin K status.