The long-range goal of this proposal is to identify the cellular and molecular signaling processes by which G protein-coupled receptors (GPCRs) modulate growth of human airway smooth muscle (ASM) cells. Numerous inflammatory and contractile agents known to activate GPCRs are elevated in the airways of patients with asthma, a disease characterized by airway hyperreactivity and increased ASM mass. The P.I.'s studies establish that despite the inability of most GPCR agonists to stimulate ASM growth by themselves, these agonists are capable of significantly increasing the proliferative effect of polypeptide growth factors. The mechanisms underlying this synergy between GPCR and receptor tyrosine kinase (RTK) activation are unknown. The central hypothesis of this proposal is that GPCRS and RTKs contribute distinct signals that can cooperate to activate p7Os6k and cause a synergistic induction of HASM growth. This hypothesis is supported by extensive preliminary data that demonstrate a prolonged, increased activation of p70s6k in ASM cells stimulated by RTK (EGF) and GPCR (thrombin, histamine, carbachol, and thromboxane) agonists. To test this hypothesis, we will examine the effects of specifically inhibiting molecules/pathways potentially upstream of p70s6k on: 1) p70s6k activity; 2) p70s6k phosphorylation; and 3) ASM growth that is stimulated by EGF, GCPR agonists, or both.
In Aim 1, the role of GPCR-dependent transmembrane signaling events (specific heterotrimeric G protein activation, membrane-delineated scaffold formation) in p70s6k activation will be analyzed primarily using molecular biology techniques to inhibit heterotrimeric G protein-, arrestin-, and focal adhesion-dependent signaling.
In Aim 2, we will target cytosolic molecules (calcium and phosphoinositides, focal adhesion kinases, Src, Akt, Akt, and PKC isoforms) in a similar manner to examine their roles in increased p70s6k activation.
In Aim 3, mutants of p70s6k will be expressed in ASM cultures for the purpose of analyzing the relative contributions of EGF and GPCR agonists in the phosphorylation of specific amino acids of p70s6k.
In Aim 4, molecules/pathways shown to mediate the GPCR-dependent modulation p70s6k activation will be inhibited in assays of RTK+GPCR-dependent cell growth. Collectively these studies will clarify the role of GPCRS as transducers of growth signaling, enhance our understanding of an important growth-regulatory molecule, and identify potential targets for therapeutic management of asthma.
