LHRH-induced tyrosine phosphorylation and MAP kinase activation in alpha T3-1 cells.

Protein tyrosine kinases regulate numerous signal transduction pathways, including those controlling cell growth [l] . Apart from the growth factor receptor tyrosine kinases, certain G proteincoupled receptors also are capable of stimulating protein tyrosine phosphorylations in their target cells [2]. Another group of protein kinases that become activated in response to various extracellular stimuli to their cell surface receptor are mitogen-activated protein kinases (MAP kinases), a group of serinehhreonine protein hnases 131. MAP kinases can be activated not only by tyrosine kinase receptors, like the EGF receptor [4], but also by G protein-linked receptors [2,5] and therefore MAP kinase may also be involved in non-proliferative signalling cascades. In this study we have investigated the possibility that the luteinising hormone-releasing hormone (LHRH) receptor, a classical phosphoinositide receptor (which is known to couple to Gq [6]), may also activate these unconventional signalling pathways (tyrosine kinases and MAP kinases) in aT3-1 cells. To enable us to investigate LHRH-induced tyrosine phosphorylations and MAP kinase (p42/p44) activation in wT31 cells we used electrophoretic and immunoblotting techniques. The aT3-1 cells were grown to confluence in 12 well plates in 1 ml DMEM containing 10% foetal calf serum at 37°C under 5% CO2. After 3-4 days cells were serum-starved for 24 hours prior to the experiment. For immunoblot analysis of tyrosine phosphorylation and the mobility shift of MAP kinase, cells were stimulated, lysed in SDS-PAGE buffer and separated on a 7.5% or 20% homogeneous microgel, respectively. After separation by electrophoresis (PhastSystem, Pharmacia), proteins were transferred to a polyvinylidene difluoride membrane. Non-specific binding by antibodies was prevented by incubating the blots in 5% BSAPBS overnight. The blots were then washed twice at room temperature in PBS/O.l% Tween-20 and incubated for 1-3 h with the polyclonal anti-phosphoTyr antibody (4G10, UBI) or the monoclonal anti-MAP kinase antiserum (2033, Zymed) in 0.25% BSAPBS. After extensive washing the blots were subsequently incubated for 1 h at room temperature with horseradish peroxide (HRP) anti-mouse IgG in 0.25% BSA/PBSnween. After washing, immunostained proteins were visualized using the Enhanced Chemiluminescence (ECL) detection system (Amersham International plc). The anti-phosphoTyr immunoblotting experiments revealed that LHRH stimulated tyrosine phosphorylation of proteins of 65-75, 125 and 130 kDa. The effect of LHRH was concentrationand timedependent, being maximal at 1-10 nM after 5-10 minutes and remaining elevated for at least 60 minutes. Furthermore LHRH was inferred to cause phosphorylation of the p42/p44 MAP kinase. This activation was detectable on anti-MAP kinase immunoblots where the activated phosphorylated forms of p42/p44 display a lower electrophoretic mobility. To investigate the possible involvement of protein kinase C (PKC) as an upstream component of this signalling cascade, we have used the selective PKC inhibitor GF109203X. Incubation with this agent (3 pM) inhibited the LHRH-induced tyrosine phosphorylation partially and prevented the MAP kinase mobility shift. Phorbol esters also caused tyrosine phosphorylations and MAP kinase activation. We also investigated the role of G proteins by exposing the aT3-1 cells to pertussis toxin (FTX, 100 ng/ml; an inhibitor of receptor coupling to Gi/Go) for 18 hours prior to the experiment. Treatment with PTX blocked the LHRH-induced MAP kinase activation without affecting the 3 major tyrosine phosphorylated bands. These findings suggest that the LHRH receptor probably acts through more than one G protein (ie Gi/Go in addition to Gq) and activates at least two independent signalling pathways. The MAP kinase activation might act through a Gi-mediated and p21ras-dependent route, as has been reported for the a2-adrenergic and lysophosphatidic acid (LPA) receptor [2,5]. However, the LHRH-induced tyrosine phosphorylations are probably associated with Gq and phospholipase C (PLC) activation, as has been shown for the LPA receptor 121. The molecular masses of the tyrosine phosphorylated bands, through the LHRH receptor, are quite similar to LPA-stimulated tyrosine phosphorylations [7,8]. The 125 kDa and 130 kDa proteins could be the focal adhesion kinase (p12SFAK) and p130, while the 65-75 kDa band might include paxillin (a possible substrate for p 12SFAK) 181. The findings suggest that both of these unconventional signalling pathways are dependent, at least in part, on ;I form of