Abstract
U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R). Substance P (SP), the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK) signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK) is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells.
Highlights
Substance P (SP) is a member of the tachykinin family of neuropeptides and it is best known as a neurotransmitter in the central and peripheral nervous systems
In the present study we show that U373MG astrocytoma cells that endogenously express the full-length neurokinin 1 receptor (NK1R) [29] respond with membrane blebbing when SP is added to medium, but the dynamics of cell morphology changes are distinct from morphological changes we have previously described in HEK293 cells expressing recombinant NK1R
U373MG cells endogenously express this receptor. Both cell types undergo morphological changes, there are differences between the changes induced by SP in U373MG and those induced in HEK293-NK1R cells
Summary
Substance P (SP) is a member of the tachykinin family of neuropeptides and it is best known as a neurotransmitter in the central and peripheral nervous systems. The effects of SP are mediated by the NK1R, a G-protein coupled receptor which is expressed in many tissues, including the nervous system, the gut, salivary glands, and cells of the immune system [5,14]. The classical NK1R has a primary structure that includes 407 amino acid residues and is coupled to proteins in the Gq family, leading to phospholipase C activation, intracellular calcium increase and PKC activation [15]. A truncated splice variant of NK1R that lacks 96 amino acid residues at the Cterminus has been described [17,18,19,20], with roles in modulation of responses triggered by cytokines, chemotaxis of macrophages and regulation of HIV replication [15]
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