Cells expressing G protein linked receptors for neurotransmitters and hormones rapidly (seconds to minutes) desensitize and gradually (minutes) resensitize after brief exposure to agonist. Continuous exposure to agonist induces a slow (hours) down-regulation of receptor number and a reduction in cellular responsiveness, which recover after agonist removal. General Hypothesis 1 is that desensitization is mediated by a) receptor endocytosis; and b) phosphorylation-mediated uncoupling of the receptor from G proteins. Resensitization requires receptor recycling and dephosphorylation. General Hypothesis 2 is that down-regulation is mediated by a) endocytosis and trafficking of the receptor to lysosomes; and b) decreased synthesis of new receptors. Recovery requires new protein synthesis. These mechanisms tightly regulate cellular responses, and their derangement leads to disease. This proposal focuses on mechanisms that attenuate responses of cells expressing the NK1 receptor (NK1-R) to stimulation by the neuropeptide substance P (SP). Many of the effects of SP in the peripheral and central nervous system are mediated by the NK1-R. Desensitization and down- regulation will be examined in a) cell lines transfected with cDNA encoding the NK1-R; b) primary cultures of myenteric neurons or an intestinal muscle cell line, cell types which naturally express the NK1-R and which are of great importance for control of digestion; and c) enteric neurons and muscle cells of the intact animal.
Specific Aim 1 examines the pathway, mechanism and functional importance of endocytosis of the NK1-R. Experiments will a) delineate the endocytic pathway using receptor antibodies and fluorescent SP, and determine whether endocytosis of the NK1-R can be used as a marker for the reflex release and site of action of SP; b) determine by mutation if the NPXXY motif in the 7th transmembrane domain of the NK1-R is required for endocytosis; and c) determine whether endocytosis is required for desensitization and resensitization using endocytosis inhibitors and internalization-defective mutant receptors.
Specific Aim 2 investigates the mechanism and functional importance of uncoupling of the NK1-R from G protein to desensitization. Experiments will a) determine the contributions of kinases and beta-arrestins to SP- induced phosphorylation and desensitization of the NK1-R using inhibitors, overexpression, and mutation of potential phosphorylation sites; and b) investigate whether kinases and beta-arrestins are appropriately localized with the NK1-R to uncouple signal transduction.
Specific Aim 3 examines the pathways, mechanisms and functional importance of down-regulation of the NK1-R. Experiments will a) examine endocytosis and trafficking of the NK1-R to lysosomes during down-regulation; b) determine whether mRNA levels for the NK1-R are also down-regulated by continuous exposure to SPO; and c) investigate the importance of domains of the NK1-R and kinases in down-regulation.
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