EGF precursor mRNA and membrane-associated EGF precursor protein in rat exorbital lacrimal gland

Abstract

This study was designed to demonstrate the presence of epidermal growth factor (EGF) in the rat exorbital lacrimal gland. EGF precursor gene transcription was demonstrated first by RT-PCR analysis of lacrimal gland RNA using a set of specific primers and second by Northern blot analysis of rat lacrimal gland mRNA. A rabbit polyclonal antibody (rEGF2) directed against rat submaxillary gland EGF was used to detect EGF-containing proteins by RIA. Results indicate that the rat lacrimal gland does not contain detectable soluble and mature EGF but that the EGF immunoreactivity is associated with the membrane-enriched fraction. Analysis of the detergent-solubilized membrane proteins by gel filtration shows that membrane-associated EGF immunoreactivity was present as a high-molecular-mass protein. Moreover, as shown by Western blot analysis, a specific anti-rat EGF precursor antibody (ppEGF1) can immunoprecipitate a 152-kDa EGF-containing protein. Taken together, these results demonstrate for the first time both EGF precursor gene transcription and EGF precursor protein expression in a lacrimal tissue, i.e., the rat exorbital lacrimal gland. The demonstration that EGF appears to be stored only as its full-length membrane precursor may provide important information to study the regulation of its secretory process.

Proprotein Convertase PC7 Enhances the Activation of the EGF Receptor Pathway through Processing of the EGF Precursor

“…However, we did find that only fulllength membrane-bound PC7 can enhance the processing of pro-EGF into an intermediate ϳ115-kDa transmembrane form (EGF-115; Fig. 1), which had already been observed in kidney, but not in lacrimal gland, extracts (12). Interestingly, we demonstrate that in kidney both PC7 and EGF mRNAs are abundant, but PC7 expression is very low in lacrimal glands (Fig.…”

“…Form-We first tested the possible implication of PCs in the processing of the cell surface pro-EGF, which may mimic the undefined kidney protease reported to be implicated in pro-EGF maturation (12). Thus, we co-expressed pro-EGF in HEK293 or Neuro2A cells with PC1/3, Furin, PC5/6A, PACE4, or PC7, the only basic aa-specific PCs that can function in the trans-Golgi network and/or cell surface (16).…”

“…In contrast, in kidney, pro-EGF is found at the cell surface mainly as an unprocessed protein (1,8) or as intermediate forms generated by unknown proteases (9 -11). Among the processed forms in kidney, a prominent ϳ115-kDa membrane-bound intermediate (herein called EGF-115) was reported (12), as well as a soluble form in urine generated by an undefined serine protease (10).…”

Regulated Cell Surface Pro-EGF Ectodomain Shedding Is a Zinc Metalloprotease-dependent Process

“…In other tissues studied so far such as kidney (12, 36 -39), mammary (40), and lacrimal glands (41), the fully mature 6 kDa EGF was undetectable, and EGF accumulated as its high molecular weight precursor associated with the apical membrane of epithelial cells. The presence of EGF-containing peptides of varying molecular masses (from 6 to 160 kDa) in urine (37,42,43) and milk (22) of different species indicates that EGF can be released (shed) from its membrane-anchored precursor.…”

“…In order to lead to the ultimate, mature 6-kDa form of the molecule, the ectodomain needs to be further maturated in the extracellular medium. It has already been shown that, in vitro, mature EGF could be released from its precursor through the action of different type of proteases; acidic protease such as pepsin (36) or serine-proteases such as trypsin (22,39,41) and plasmin (44). Moreover, membrane fractions of both kidney (45,46) and mammary gland (47) contain a membrane-bound proteolytic activity that colocalized with pro-EGF and released soluble EGF from its membraneassociated precursor.…”