A functional fluorescent neurokinin NK2 receptor was constructed by joining enhanced green fluorescent protein to the amino-terminal end of the rat NK2 receptor and was expressed in human embryonic kidney cells. On cell suspensions, the binding of fluorescent Bodipy-labeled neurokinin A results in a saturatable and reversible decrease of NK2 receptor fluorescence via fluorescence resonance energy transfer. This can be quantified for nM to M agonist concentrations and monitored in parallel with intracellular calcium responses. On single cells, receptor site occupancy and local agonist concentration can be determined in real time from the decrease in receptor fluorescence. Simultaneous measurement of intracellular calcium responses and agonist binding reveals that partial receptor site occupancy is sufficient to desensitize cellular response to a second agonist application to the same membrane area. Subsequent stimulation of a distal membrane area leads to a second response to agonist, provided that it had not been exposed to agonist during the first application. Together with persistent translocation of fluorescent protein kinase C to the membrane area exposed to agonist, the present data support that not only homologous desensitization but also heterologous desensitization of NK2 receptors is compartmentalized to discrete membrane domains.Neurotransmitters stimulate target cells upon interacting with two major categories of regulatory proteins, ligand-gated ion channels (1-3), and G protein-coupled receptors (4). For ligand-gated ion channels, response activation and termination are mediated by a single molecule that carries the neurotransmitter site and the effector site (1). In contrast, responses mediated by G protein-coupled receptors result from a complex cascade of transient and sequential interactions between the receptor and a series of distinct messengers and effector proteins, which belong to distinct subcellular compartments (5-9).Tachykinins or neurokinins form a family of related neuropeptides found throughout central and peripheral nervous tissues. Their biological activities related to pain transmission (10, 11), smooth muscle contraction, vasodilation, or neurogenic inflammation are mediated by at least three distinct G protein-coupled receptors, NK1, NK2, and NK3, that show preferential binding for the endogenous agonists substance P, neurokinin A (NKA), 1 and neurokinin B (NKB), respectively (12). All three receptor isotypes belong to the family of seven transmembrane regulatory proteins and are coupled to an intracellular calcium release response primarily mediated by the pertussis toxin-insensitive heterotrimeric GTP-binding protein Gq/G11.In the present work, we have used fluorescence resonance energy transfer-based detection of neurokinin A binding to its G protein-coupled receptor, the NK2 tachykinin receptor, to detect real-time interactions on living cells and to study the spatial distribution of response activation and desensitization at the single cell level. Tachykinin NK2 receptors w...

Abstractp53, a major sensor of DNA damage, is a transcription factor that, depending on its phosphorylation status, regulates the cell cycle, DNA repair, or apoptosis. The protein kinase C (PKC) family of isozymes is also implicated in cell cycle and programmed cell death (PCD) control and has recently been shown to influence p53 function. Using three human colon adenocarcinoma cell lines SW480, EB-1, and HCT116 that either lack p53 function and were engineered to express inducible wild-type p53 (wt p53), or that constitutively express wt p53, we show that phorbol ester-mediated PKC activation potentiates p53-induced PCD. Despite the effectiveness of PKC/p53 synergy in inducing SW480 tumor cell death, however, a fraction of the cells invariably survive. To address the putative mechanisms that underlie resistance to PKC/p53-induced cell death, we generated a phorbol 12-myristate 13-acetate/p53-resistant SW480 subline and compared the gene expression profile of resistant and parental cells by DNA microarray analysis. The results of these experiments show that PKC/p53-resistant cells express a higher level of several matrix metalloproteinases (MMP), including MMP-9, MMP-10, and MMP-12, and corresponding real-time PCR assays indicate that p53 is a negative regulator of MMP-9 gene expression. Using MMP inhibitors and MMP-specific small interfering RNA, we show that MMP function confers protection from PKC/p53-induced apoptosis and identify the protective MMPs as MMP-9 and MMP-10. Taken together, these observations provide evidence that MMPs are implicated in tumor cell resistance to the synergistic proapoptotic effect of PKC and p53. (Cancer Res 2005; 65(10): 4261-72)

A functional fluorescent neurokinin NK2 receptor, EGFP-NK2, was previously used to follow, by fluorescence resonance energy transfer measurements in living cells, the binding of its fluorescently labeled agonist, bodipy-neurokinin A (NKA). Local agonist application suggested that the activation and desensitization of the NK2 receptors were compartmentalized at the level of the plasma membrane. In this study, fluorescence recovery after photobleaching experiments are carried out at variable observation radius (vrFRAP) to probe EGFP-NK2 receptor mobility and confinement. Experiments are carried out at 20°C to maintain the number of receptors constant at the cell surface during recordings. In the absence of agonist, 35% EGFP-NK2 receptors diffuse within domains of 420 ؎ 80 nm in radius with the remaining 65% of receptors able to diffuse with a long range lateral diffusion coefficient between the domains. When cells are incubated with a saturating concentration of NKA, 30% EGFP-NK2 receptors become immobilized in small domains characterized by a radius equal to 170 ؎ 50 nm. Biochemical experiments show that the confinement of EGFP-NK2 receptor is not due to its association with rafts at any given time. Colocalization of the receptor with ␤-arrestin and transferrin supports that the small domains, containing 30% of activated EGFP-NK2, correspond to clathrin-coated pre-pits. The similar amount of confined EGFP-NK2 receptors found before and after activation (30 -35%) is discussed in term of putative transient interactions of the receptors with preexisting scaffolds of signaling molecules.The general mechanisms underlying the activation of Gprotein-coupled receptors (GPCRs) 1 and the following attenuation of the cellular response seem well established at the molecular level. The prevailing view is that the intracellular signal results from a cascade of transient and sequential molecular interactions starting with the activation of the heterotrimeric G-protein upon ligand/receptor interaction. The biological effect then can be attenuated by three main mechanisms: (i) agonists removal; (ii) agonist-mediated desensitization of the receptor molecules by uncoupling of the activated receptors from heterotrimeric G-proteins; and (iii) agonist-stimulated receptor endocytosis followed by either recycling or degradation. The remaining key questions are to understand the architecture governing the molecular interactions of the signaling cascade and how these multiple interactions take place in space and time, notably starting with the lateral organization of the partners at the level of the plasma membrane (1-4). Here we investigate these different aspects using the neurokinin NK2 receptor expressed in HEK293 cells as a model system.Neurokinins form a family of neuropeptides acting at three distinct GPCRs, namely NK1, NK2 and NK3. The NK2 receptor shows preferential binding for the endogenous neurokinin A (NKA) (5) and is mostly found in peripheral nervous tissues where it participates mainly to smooth muscle contraction. In pr...

Any list of past and recent findings on vertebrate brain prenatal development would have to include the fundamental roles of homeobox genes, the genes encoding the nuclear regulatory homeodomain proteins. The discovery of homeobox genes and their involvement as master regulatory elements in programing the development of an embryo into a complete adult organism has provided a key to our understanding of ontogenesis. Also, the correlation of mouse developmental mutants and their corresponding human syndromes with mutations in homeobox genes has provided further evidence for the fundamental role of homeobox genes during the vertebrate brain embryonic development. Here, we review the expression patterns and the phenotypes of gene mutations that implicate a large repertoire of mouse homeobox genes in the specification of neuronal functions during brain embryogenesis.