Characterization of a membrane tyrosine phosphatase in AR42J cells: regulation by somatostatin

Abstract

A phosphotyrosyl protein phosphatase (PTPase) activity has been characterized in the plasma membranes of confluent AR42J pancreatic tumor cells using 32P-labeled poly(Glu, Tyr) as substrate. Membrane PTPase activity exhibited an apparent Michaelis constant of 3 microM and an apparent maximal velocity of 0.9 nmol.min-1.mg-1. It was inhibited by orthovanadate, zinc, poly(Glu,Tyr) and was stimulated by EDTA and dithiothreitol. Gel filtration of solubilized plasma membranes gave a peak of enzyme activity at a relative molecular weight of 70,000. Plasma membrane PTPase activity was changed during AR42J cell growth. At the beginning of culture, the control PTPase activity was minimal. Over the 5 days of culture, PTPase activity increased to reach a maximum (3.5-fold over control activity) preceding confluency by 2 days. Then the high level of PTPase activity was sustained until confluency. Incubation of the cells with the stable somatostatin analogue SMS 201-995 (SMS) resulted in a rapid and transient activation of crude membrane PTPase activity. Activation reached a maximum level within 5 min of addition and return to control levels within 20 min. The effect of SMS was dose dependent with half-maximal and maximal activation occurring at 6 pM and 0.1 nM SMS respectively.

Induction of a negative autocrine loop by expression of sst2 somatostatin receptor in NIH 3T3 cells.

“…5 A). As previously observed for tyrosine phosphatase activity in pancreatic cells (7), in control and sst2-transfected cells, PTP1C activity decreased when NIH3T3 cells reached confluency, after 4-5 d of culture. In these conditions, PTP1C activity was increased by ‫ف‬ 27% (P Ͻ 0.05) in cells expressing sst2 (0.19Ϯ0.008 pmol/ mg protein/min), as compared to that of control cells (0.15Ϯ0.006 pmol/mg protein/min, n ϭ 3).…”

“…We have established that cells expressing sst2 were capable of producing somatostatin 14 in cell media at concentration (0.2-0.3 pM) high enough to trigger a slight inhibitory growth response (7,(20)(21). However, due to the well known short half-life of the peptide in body fluids as in cell culture medium (2), the detected somatostatin 14 concentration may reflect only a fraction of the really secreted peptide.…”

“…In pancreatic tumor cells, we and others, have demonstrated that analogues of somatostatin antagonize the mitogenic effect of growth factors acting on tyrosine kinase receptors such as epidermal growth factor and fibroblast growth factor (12,13). Furthermore, these analogues cause the stimulation of a membrane tyrosine phosphatase that may be involved in the intracellular mechanism of somatostatin-induced growth inhibition (7,12,14).…”

“…The biological effect of somatostatin is mediated by G-protein coupled receptors that mediate a variety of signal transduction pathways including adenylate cyclase, ionic conductance channels and protein dephosphorylation (3)(4)(5)(6)(7).…”

Distribution and second messenger coupling of four somatostatin receptor subtypes expressed in brain

“…The expression of the SMS 201-995 sensitive receptor subtype SSTRZ in the hippocampus suggests that the anticonvulsant actions of SRIF may be mediated through SSTR2. SRIF receptors which have been shown to belong to the family of G-protein coupled receptors, act on various cellular effector systems such as adenylyl cyclases [14], ion channels f4] or phosphatases [45]. In order to characterize the cellular effector systems of the cloned SSTR subtypes, human SSTRl-4 were stably expressed in HEK 293 cells.…”