A novel mechanism for isoprenaline-stimulated proliferation of rat parotid acinar cells involving the epidermal growth factor receptor and cell surface galactosyltransferase

Abstract

Chronic injections of epidermal growth factor (EGF) or the beta-adrenergic receptor agonist isoprenaline resulted in rat parotid gland hypertrophy and hyperplasia. Introduction of a polyclonal antibody to EGF or the EGF-receptor (EGF-R) caused a specific retardation of acinar cell proliferation when injected along with the growth factor. Meanwhile, only the antibody to EGF-R caused a dose-dependent retardation of proliferation on co-administration with isoprenaline both in vivo and in vitro. The antibody injected alone had no effect on cell growth. When cells were incubated in the presence of EGF, plasma membranes from isoprenaline-treated and control animals showed phosphorylation of the EGF-R tyrosine moieties and transient increases in membrane-associated phospholipase C gamma. Isoprenaline did not stimulate phosphorylation of the EGF-R in isolated plasma membranes. However, activation of the phosphotyrosine-signalling pathway could be duplicated by incubating isoprenaline-treated acinar cells, but not control cells, with bovine galactosyltransferase. Immunopurified EGF-R demonstrated variations in reactivity with two different lectins after treatment of the cells with the beta-agonist as well as increased galactosyltransferase substrate capacity in vitro. In addition, incubation of intact acinar cells and isolated plasma-membrane fractions from isoprenaline-treated rats with UDP-[14C]galactose resulted in an increased incorporation of label into the EGF-R. The results suggest that the carbohydrate moiety of the EGF-R has been altered in isoprenaline-treated animals allowing galactosyltransferase now to recognize this receptor. This interaction may in part mediate proliferation of parotid acinar cells. Indeed, we have previously shown that an antibody to galactosyltransferase is capable of blocking isoprenaline-mediated acinar cell proliferation in vivo [Humphreys-Beher, Schneyer, Kidd& Marchase (1987) J. Biol. Chem. 262, 11706-11713].

Rat Parotid Gland Acinar Cell Proliferation: Signal Transduction at the Plasma Membrane

“…Following 3 d of twice-daily injections, the animals were killed and gland hypertrophy and hyperplasia determined. Gland wet weight was partially reduced in a dose-dependent fashion (Purushotham et al, 1992). The anti-EGF-R serum had no growth stimulating effect as its own.…”

“…In vivo appearance of cell-surface Gal Tase begins by 3 h following a single injection of ISO (Marchase et al, 1988) hydrate changes. The parallel appearance of the transferase in the membrane along with changes in the structure of the oligosaccharide sidechains of the EGF-R support the idea of Gal Tase stimulation of growth by a "receptor-ligand-type" interaction (HumphreysBeher, 1989;Purushotham et al, 1992).…”

“…Detection of altered oligosaccharide structures for the EGF-R were determined by the use of biotinylated lectins with various carbohydrate specificities. An increased lectin binding for the EGF-R following ISO treatment was observed with WGA and coral tree (CorT) lectins that are specific for GlcNAc and polylactosamines, respectively (Purushotham et al, 1992). Consistent with the increased binding of WGA and CorT lectins, the purified receptor from ISO-treated animals acted as a better substrate in an in vitro galactosylation assay than the control protein (0.038 nmol Gal incorp/lh vs. 0.004 nmol Gal incorp/h using 0.5gg receptor protein).…”

“…Furthermore, a variety of agents that interact with cell-surface Gal Tase interfere with the generation of the cellular signals for growth in this tissue so as to inhibit growth (Humphreys-Beher et al, 1987 Although we have previously reported the presence of the epidermal growth factor-receptor (EGF-R) in acinar cells (Humphreys-Beher et al, 1990) as well as the fact that following ISO treatment, a 170 kDa plasma membrane protein becomes tyrosine phosphorylated, it was of interest to determine if the receptor was, indeed, a substrate for galactosylation by bovine and endogenous Gal Tase. Plasma membrane proteins were therefore immune precipitated following 14C-labeling using a polyclonal antibody to rat EGF-R (Purushotham et al, 1992). As revealed in Figure 1 treatment contained three-to fivefold higher levels of ['4C]-Gal than that isolated from an equal quantity of membrane protein (nontreated) following immunoprecipitation (Purushotham et al, 1992).…”

“…Plasma membrane proteins were therefore immune precipitated following 14C-labeling using a polyclonal antibody to rat EGF-R (Purushotham et al, 1992). As revealed in Figure 1 treatment contained three-to fivefold higher levels of ['4C]-Gal than that isolated from an equal quantity of membrane protein (nontreated) following immunoprecipitation (Purushotham et al, 1992). In a further attempt to characterize the possible alteration in the carbohydrate moieties contributing to an increase in the ability of this glycoprotein to act as a Gal Tase substrate, the EGF-R was purified by immunoaffinity chromatography.…”