Glutamate involvement in calcium-dependent migration of astrocytoma cells.

BACKGROUND: Astrocytoma are known to have altered glutamate machinery that results in the release of large amounts of glutamate into the extracellular space but the precise role of glutamate in favoring cancer processes has not yet been fully established. Several studies suggested that glutamate might provoke active killing of neurons thereby producing space for cancer cells to proliferate and migrate. Previously, we observed that calcium promotes disassembly of integrin-containing focal adhesions in astrocytoma, thus providing a link between calcium signaling and cell migration. The aim of this study was to determine how calcium signaling and glutamate transmission cooperate to promote enhanced astrocytoma migration. METHODS: The wound-healing model was used to assay migration of human U87MG astrocytoma cells and allowed to monitor calcium signaling during the migration process. The effect of glutamate on calcium signaling was evaluated together with the amount of glutamate released by astrocytoma during cell migration. RESULTS: We observed that glutamate stimulates motility in serum-starved cells, whereas in the presence of serum, inhibitors of glutamate receptors reduce migration. Migration speed was also reduced in presence of an intracellular calcium chelator. During migration, cells displayed spontaneous Ca(2+) transients. L-THA, an inhibitor of glutamate re-uptake increased the frequency of Ca(2+) oscillations in oscillating cells and induced Ca(2+) oscillations in quiescent cells. The frequency of migration-associated Ca(2+) oscillations was reduced by prior incubation with glutamate receptor antagonists or with an anti-ß1 integrin antibody. Application of glutamate induced increases in internal free Ca(2+) concentration ([Ca(2+)]i). Finally we found that compounds known to increase [Ca(2+)]i in astrocytomas such as thapsigagin, ionomycin or the metabotropic glutamate receptor agonist t-ACPD, are able to induce glutamate release. CONCLUSION: Our data demonstrate that glutamate increases migration speed in astrocytoma cells via enhancement of migration-associated Ca(2+) oscillations that in turn induce glutamate secretion via an autocrine mechanism. Thus, glutamate receptors are further validated as potential targets for astrocytoma cancer therapy.

FAK phosphorylation at Tyr-925 regulates cross-talk between focal adhesion turnover and cell protrusion.

Cell migration is a highly complex process that requires the coordinated formation of membrane protrusion and focal adhesions (FAs). Focal adhesion kinase (FAK), a major signaling component of FAs, is involved in the disassembly process of FAs through phosphorylation and dephosphorylation of its tyrosine residues, but the role of such phosphorylations in nascent FA formation and turnover near the cell front and in cell protrusion is less well understood. In the present study, we demonstrate that, depending on the phosphorylation status of Tyr-925 residue, FAK modulates cell migration via two specific mechanisms. FAK⁻/⁻ mouse embryonic fibroblasts (MEFs) expressing nonphosphorylatable Y925F-FAK show increased interactions between FAK and unphosphorylated paxillin, which lead to FA stabilization and thus decreased FA turnover and reduced cell migration. Conversely, MEFs expressing phosphomimetic Y925E-FAK display unchanged FA disassembly rates, show increase in phosphorylated paxillin in FAs, and exhibit increased formation of nascent FAs at the cell leading edges. Moreover, Y925E-FAK cells present enhanced cell protrusion together with activation of the p130(CAS)/Dock180/Rac1 signaling pathway. Together, our results demonstrate that phosphorylation of FAK at Tyr-925 is required for FAK-mediated cell migration and cell protrusion.

Contribution of a tyrosine-based motif to cellular trafficking of wild-type and truncated NPY Y(1) receptors.

The human NPY Y(1) receptor undergoes fast agonist-induced internalization via clathrin-coated pits then recycles back to the cell membrane. In an attempt to identify the molecular determinants involved in this process, we studied several C-terminal truncation mutants tagged with EFGP. In the absence of agonist, Y(1) receptors lacking the last 32 C-terminal amino acids (Y(1)Δ32) are constitutively internalized, unlike full-length Y(1) receptors. At steady state, internalized Y(1)Δ32 receptors co-localize with transferrin, a marker of early and recycling endosomes. Inhibition of constitutive internalization of Y(1)Δ32 receptors by hypertonic sucrose or by co-expression of Rab5aS34N, a dominant negative form of the small GTPase Rab5a or depletion of all three isoforms of Rab5 indicates the involvement of clathrin-coated pits. In contrast, a truncated receptor lacking the last 42 C-terminal amino acids (Y(1)Δ42) does not constitutively internalize, consistent with the possibility that there is a molecular determinant responsible for constitutive internalization located in the last 10 amino acids of Y(1)Δ32 receptors. We show that the agonist-independent internalization of Y(1)Δ32 receptors involves a tyrosine-based motif YXXΦ. The potential role of this motif in the behaviour of full-length Y(1) receptors has also been explored. Our results indicate that a C-terminal tyrosine-based motif is critical for the constitutive internalization of truncated Y(1)Δ32 receptors. We suggest that this motif is masked in full-length Y(1) receptors which do not constitutively internalize in the absence of agonist.

Activation of CD47 receptors causes proliferation of human astrocytoma but not normal astrocytes via an Akt-dependent pathway.

CD47 is a membrane receptor that plays pivotal roles in many pathophysiological processes, including infection, inflammation, cell spreading, proliferation, and apoptosis. We show that activation of CD47 increases proliferation of human U87 and U373 astrocytoma cells but not normal astrocytes. CD47 function-blocking antibodies inhibit proliferation of untreated U87 and U373 cells but not normal astrocytes, suggesting that CD47 may be constitutively activated in astrocytoma. CD47 expression levels were similar in our three cell types. CD47 couples to G-proteins in astrocytes and astrocytoma and especially to the Gßγ dimer. Downstream signaling following CD47 activation involves Gßγ dimer-dependent activation of the PI3K/Akt pathway in astrocytoma cells but not in normal astrocytes. This pathway is known to be deregulated in astrocytoma, leading to cell proliferation and enhanced survival signals. Putative PLIC-1 interaction with CD47 in astrocytoma cells but not astrocytes may contribute to the proliferative effect observed upon activation of CD47. Our data indicate that CD47 receptors have a stimulatory role in cell proliferation and demonstrate for the first time that CD47 signals via the PI3K/Akt pathway in cancerous cells but not normal cells.

Hyperphosphorylated FAK Delocalizes from Focal Adhesions to Membrane Ruffles.

Cell adhesion and migration are key determinants in tumor metastasis. Adherence of tumor cell to the extracellular matrix is mediated via integrin containing focal adhesions (FAs). Binding of integrins to ECM triggers phosphorylation of two major components of FAs, focal adhesion kinase (FAK) and Src, activating downstream signaling pathway which leads to FA disassembly and cell migration. In this paper, we analyze how phosphorylation of FAK regulates its trafficking at FAs in living human astrocytoma cells. Upon pervanadate-induced FAK phosphorylation, phosphorylated FAK appeared highly expressed at newly formed membrane ruffles. This effect was abolished in presence of the specific Src inhibitor PP2. Our findings demonstrate that upon phosphorylation, FAK delocalizes from FAs to membrane ruffles.

Distinct motifs of neuropeptide Y receptors differentially regulate trafficking and desensitization.

Activated human neuropeptide Y Y(1) receptors rapidly desensitize and internalize through clathrin-coated pits and recycle from early and recycling endosomes, unlike Y(2) receptors that neither internalize nor desensitize. To identify motifs implicated in Y(1) receptor desensitization and trafficking, mutants with varying C-terminal truncations or a substituted Y(2) C-terminus were constructed. Point mutations of key putative residues were made in a C-terminal conserved motif [phi-H-(S/T)-(E/D)-V-(S/T)-X-T] that we have identified and in the second intracellular i2 loop. Receptors were analyzed by functional assays, spectrofluorimetric measurements on living cells, flow cytometry, confocal imaging and bioluminescence resonance energy transfer assays for beta-arrestin activation and adaptor protein (AP-2) complex recruitment. Inhibitory GTP-binding protein-dependent signaling of Y(1) receptors to adenylyl cyclase and desensitization was unaffected by C-terminal truncations or mutations, while C-terminal deletion mutants of 42 and 61 amino acids no longer internalized. Substitutions of Thr357, Asp358, Ser360 and Thr362 by Ala in the C-terminus abolished both internalization and beta-arrestin activation but not desensitization. A Pro145 substitution by His in an i2 consensus motif reported to mediate phosphorylation-independent recruitment of beta-arrestins affected neither desensitization, internalization or recycling kinetics of activated Y(1) receptors nor beta-arrestin activation. Interestingly, combining Pro145 substitution by His and C-terminal substitutions significantly attenuates Y(1) desensitization. In the Y(2) receptor, replacement of His155 with Pro at this position in the i2 loop motif promotes agonist-mediated desensitization, beta-arrestin activation, internalization and recycling. Overall, our results indicate that beta-arrestin-mediated desensitization and internalization of Y(1) and Y(2) receptors are differentially regulated by the C-terminal motif and the i2 loop consensus motif.

Fluorescent probe based on intramolecular proton transfer for fast ratiometric measurement of cellular transmembrane potential.

Development of fast-response potentiometric probes for measuring the transmembrane potential Vm in cell plasma membranes remains a challenge. To overcome the limitations of the classical charge-shift potentiometric probes, we selected a 3-hydroxychromone fluorophore undergoing an excited-state intramolecular proton transfer (ESIPT) reaction that generates a dual emission highly sensitive to electric fields. To achieve the highest sensitivity to the electric field associated to Vm, we modified the fluorophore by adding two rigid legs containing terminal polar sulfonate groups to allow a deep vertical insertion of the fluorophore into the membrane. Fluorescence spectra of the new dye in lipid vesicles and cell membranes confirm the fluorophore location in the hydrophobic region of the membranes. Variation of Vm in lipid vesicles and cell plasma membranes results in a change of the intensity ratio of the two emission bands of the probe. The ratiometric response of the dye in cells is approximately 15% per 100 mV, and is thus quite large in comparison with most single-fluorophore, fast-response probes reported to date. Combined patch-clamp/fluorescence data further show that the ratiometric response of the dye in cells is faster than 1 ms. Analysis of the excitation and emission shifts further suggests that the probe responds to changes in Vm by a mechanism based on electrochromic modulation of its ESIPT reaction. Thus, for the first time, the ESIPT reaction has been successfully applied as a sensing principle for detection of transmembrane potential, allowing to couple classical electrochromic band shifts with changes in the relative intensities of the two well-separated emission bands. The fast two-band ratiometric response as well as the relatively high sensitivity of the new probe are the key features that make it useful for rapid detection of Vm changes in cell suspensions and single cells. Moreover, the new design principles proposed in the present work should allow further improvement of the probe sensitivity.

The small alpha5beta1 integrin antagonist, SJ749, reduces proliferation and clonogenicity of human astrocytoma cells.

The potential role of alpha5beta1 integrins in cancer has recently attracted much interest. However, few alpha5beta1-selective antagonists have been developed compared with other integrins. The most specific nonpeptidic alpha5beta1 antagonist described thus far, SJ749, inhibits angiogenesis by affecting adhesion and migration of endothelial cells. We investigated the effects of SJ749 in two human astrocytoma cell lines, A172 and U87, which express different levels of alpha5beta1. SJ749 dose-dependently inhibited adhesion of both cell types on fibronectin. Application of SJ749 to spread cells led to formation of nonadherent spheroids for A172 cells but had no effect on U87 cell morphology. SJ749 also reduced proliferation of A172 cells due to a long lasting G0-G1 arrest, whereas U87 cells were only slightly affected. However, under nonadherent culture conditions (soft agar), SJ749 significantly reduced the number of colonies formed only by U87 cells. As U87 cells express more alpha5beta1 than A172 cells, we specifically examined the effect of SJ749 on A172 cells overexpressing alpha5. Treatment of alpha5-A172 cells with SJ749 decreased colony formation similarly to that observed in U87 cells. Therefore, in nonadherent conditions, the effect of SJ749 on tumor cell growth characteristics depends on the level of alpha5beta1 expression. Our study highlights the importance of alpha5beta1 as an anticancer target and shows for the first time that a small nonpeptidic alpha5beta1-specific antagonist affects proliferation of tumor cells.

Regulation of focal adhesion dynamics and disassembly by phosphorylation of FAK at tyrosine 397.

One of the major tyrosine phosphorylation activities linked to integrin signalling is that of focal adhesion kinase (FAK). High amounts of FAK are located at specialised subcellular compartments known as focal adhesions. FAK tyrosine phosphorylation at focal adhesions is increased by various stimuli including integrin engagement during migration processes, growth factors and oncogene transformation. Phosphorylation of FAK at various tyrosine residues regulates focal adhesion turnover by mechanisms that are not well understood. We made a fluorescent FAK mutant (Y397F-FAK/YCam) to analyse, in living cells, how phosphorylation of FAK regulates the turnover of focal adhesions. We found that expression of Y397F-FAK/YCam in human astrocytoma cells decreases the level of phosphorylation of FAK at endogenous Tyr-397 residues and at both endogenous and exogenous Tyr-576 residues, in the putative activation loop of the kinase. This corresponds to a decrease in phosphorylation of FAK at focal adhesions in Y397F-FAK/YCam cells, since the cellular localisation of FAK phosphoTyr-576 in cells expressing Y397F-FAK/YCam or FAK/YCam was not different. Furthermore, FRAP analysis showed that phosphorylation of FAK at Tyr-397 increases specifically the time-residency of FAK at focal adhesions but not in cytosol. This in turn induces disassembly of focal adhesions at the cell tail and promotes cell motility as shown by the decrease in microtubule-mediated turnover of Y397F-FAK/YCam-containing focal adhesions. Our data show that phosphorylation of FAK at Tyr-397 is a key determinant of how FAK controls focal adhesion turnover.

3H-Noradrenaline release from mouse iris-ciliary body: role of presynaptic muscarinic heteroreceptors.

Sympathetic neurotransmitter release and its modulation by presynaptic muscarinic heteroreceptors were studied in mouse iris-ciliary bodies. Tissue preparations were preincubated with (3)H-noradrenaline and then superfused and stimulated electrically. Firstly, experimental conditions were defined, allowing study of presynaptic sympathetic inhibition in mouse iris-ciliary body. If tissue was stimulated four times with 36 pulses/3 Hz, tritium overflow peaks were reliably and reproducibly measured. As expected, these stimulation conditions led to marked alpha(2)-autoinhibition as indicated by the release-enhancing effect of the alpha(2)-antagonists phentolamine and rauwolscine. To ensure autoinhibition-free (3)H-noradrenaline release, which is optimal for studying presynaptic sympathetic inhibition, alpha(2)-receptors were blocked in all subsequent experiments. Under these conditions, evoked tritium overflow was almost completely abolished in the presence of the sodium channel blocker tetrodotoxin, indicating a neuronal origin of (3)H-noradrenaline release. Secondly, muscarinic inhibition of (3)H-noradrenaline release was characterized using the conditions described above (36 pulses/3 Hz; phentolamine 1 muM and rauwolscine 1 muM throughout). The muscarinic receptor agonist oxotremorine M decreased evoked tritium overflow in a concentration-dependent manner with an IC(50) of 0.33 muM and maximal inhibition of 51%. The concentration-response curve of oxotremorine M was shifted to the right by the muscarinic antagonists ipratropium and methoctramine, whereas pirenzepine was ineffective. The observed rank order of antagonist potencies, ipratropium > methoctramine > pirenzepine, which is typical for the M(2) subtype, indicates that presynaptic muscarinic receptors on sympathetic axons of mouse iris-ciliary bodies are predominantly M(2). Finally, inhibition of (3)H-noradrenaline release by endogenously secreted acetylcholine was investigated. Longer pulse trains, 120 pulses/3 Hz and 600 pulses/5 Hz, were used and the cholinesterase inhibitor physostigmine was added to the superfusion medium to increase synaptic levels of endogenous acetylcholine. Under these conditions, ipratropium approximately doubled the evoked overflow of tritium, indicating that endogenously released acetylcholine can activate presynaptic muscarinic heteroreceptors. In conclusion, the present experiments establish measurement of the electrically induced release of (3)H-noradrenaline from mouse iris-ciliary bodies. As in other species, noradrenaline release in this preparation was subject to presynaptic muscarinic inhibition. Our results also indicate that the presynaptic muscarinic receptors on sympathetic axons in mouse iris-ciliary body are predominantly M(2). Moreover, these receptors can be activated by both exogenous agonists and endogenously released acetylcholine and, hence, may operate physiologically in the interplay between the parasympathetic and sympathetic nervous system.

Calcium rises locally trigger focal adhesion disassembly and enhance residency of focal adhesion kinase at focal adhesions.

Focal adhesion kinase (FAK) activity and Ca(2+) signaling led to a turnover of focal adhesions (FAs) required for cell spreading and migration. We used yellow Cameleon-2 (Ycam), a fluorescent protein-based Ca(2+) sensor fused to FAK or to a FAK-related non-kinase domain, to measure simultaneously local Ca(2+) variations at FA sites and FA dynamics. Discrete subcellular Ca(2+) oscillators initiate both propagating and abortive Ca(2+) waves in migrating U87 astrocytoma cells. Ca(2+)-dependent FA disassembly occurs when the Ca(2+) wave reaches individual FAs, indicating that local but not global Ca(2+) increases trigger FA disassembly. An unexpectedly rapid flux of FAK between cytosolic and FA compartments was revealed by fluorescence recovery after photobleaching studies. The FAK-Ycam recovery half-time (17 s) at FAs was slowed (to 29 s) by Ca(2+) elevation. FAK-related non-kinase domain-Ycam had a faster, Ca(2+)-insensitive recovery half-time (11 s), which is consistent with the effect of Ca(2+) on FAK-Ycam dynamics not being due to a general modification of the dynamics of FA components. Because FAK association at FAs was prolonged by Ca(2+) and FAK autophosphorylation was correlated to intracellular Ca(2+) levels, we propose that local Ca(2+) elevations increase the residency of FAK at FAs, possibly by means of tyrosine phosphorylation of FAK, thereby leading to increased activation of its effectors involved in FA disassembly.

A fluorescence cell biology approach to map the second integrin-binding site of talin to a 130-amino acid sequence within the rod domain.

The cytoskeletal protein talin, which provides a direct link between integrins and actin filaments, has been shown to contain two distinct binding sites for integrin beta subunits. Here, we report the precise delimitation and a first functional analysis of the talin rod domain integrin-binding site. Partially overlapping cDNAs covering the entire human talin gene were transiently expressed as DsRed fusion proteins in Chinese hamster ovary cells expressing alpha(IIb)beta(3), linked to green fluorescent protein (GFP). Two-color fluorescence analysis of the transfected cells, spread on fibrinogen, revealed distinct subcellular staining patterns including focal adhesion, actin filament, and granular labeling for different talin fragments. The rod domain fragment G (residues 1984-2344), devoid of any known actin- or vinculin-binding sites, colocalized with beta(3)-GFP in focal adhesions. Direct in vitro interaction of fragment G with native platelet integrin alpha(IIb)beta(3) or with the recombinant wild type, but not the Y747A mutant beta(3) cytoplasmic tail, linked to glutathione S-transferase, was demonstrated by surface plasmon resonance analysis and pull-down assays, respectively. Here, we demonstrate for the first time the in vivo relevance of this interaction by fluorescence resonance energy transfer between beta(3)-GFP and DsRed-talin fragment G. Further in vitro pull-down studies allowed us to map out the integrin-binding site within fragment G to a stretch of 130 residues (fragment J, residues 1984-2113) that also localized to focal adhesions. Finally, we show by a cell biology approach that this integrin-binding site within the talin rod domain is important for beta(3)-cytoskeletal interactions but does not participate in alpha(IIb)beta(3) activation.

Functional maturation of nicotinic acetylcholine receptors as an indicator of murine muscular differentiation in a new nerve-muscle co-culture system.

Under normal conditions in situ, muscle fibers and motoneurons, the main partners of motor units, are strongly dependent on each other. This interdependence hinders ex vivo studies of neuromuscular disorders where nervous or muscular components are considered separately. To allow in vitro access to complex nerve-muscle relationships, we developed a novel nerve-muscle co-culture system where mouse muscle innervation is assured by rat spinal cord explants. The degree of muscular maturation during co-culture was evaluated using the distribution of nicotinic acetylcholine receptors (AChRs) and their electrophysiological characteristics before and after innervation. In myotubes from non-innervated cultures, AChRs were diffusely distributed over the entire myotube surface. Their single-channel conductance (33.5+/-0.6 pS) and mean open time (8.1+/-0.7 ms) are characteristic of AChRs described in embryonic or denervated skeletal muscles. In innervated muscle fibers from co-cultures, AChRs appear as discrete aggregates and co-localize with synaptotagmin. In addition to the embryonic type currents, in innervated fibers AChR currents having high conductance (53.3+/-5.9 pS) and short mean open time (2.6+/-0.1 ms), characteristic of AChRs at mature neuromuscular junctions, were observed. Our data support the use of this new nerve-muscle co-culture system as a reliable model for the study of murine muscular differentiation and function.

FAK-mediated inhibition of vascular smooth muscle cell migration by the tetraspanin CD9.

Migration of vascular smooth muscle cells (SMC) towards the intima is a key event in vascular proliferative diseases. We investigated a potential role for the tetraspanin CD9 in this process in a wound migration assay. Aortic SMC from CD9 knock-out mice had higher migration rates and the presumably stimulatory anti-CD9 antibody ALMA-1 inhibited migration of human SMC. The signaling pathways responsible for this inhibitory effect were investigated. In migrating CD9-/- SMC, stress fiber formation was decreased and focal adhesions were smaller and more diffusely distributed, consistent with an inhibition of integrin clustering. In migrating mouse SMC expressing CD9, focal adhesion kinase (FAK) tyrosine phosphorylation was doubled. No differences in intracellular calcium signaling were observed between CD9+/+ and CD9-/- SMC during migration. We suggest that CD9 in hibits SMC migration by a stimulation of both stress fiber formation and integrin clustering, leading to a stimulation of FAK phosphorylation.

Calcium oscillations trigger focal adhesion disassembly in human U87 astrocytoma cells.

Integrin-associated intracellular Ca(2+) oscillations modulate cell migration, probably by controlling integrin-mediated release of the cell rear during migration. Focal adhesion kinase (FAK), via its tyrosine phosphorylation activity, plays a key role in integrin signaling. In human U87 astrocytoma cells, expression of the dominant negative FAK-related non-kinase domain (FRNK) inhibits the Ca(2+)-sensitive component of serum-dependent migration. We investigated how integrin-associated Ca(2+) signaling might be coupled to focal adhesion (FA) dynamics by visualizing the effects of Ca(2+) spikes on FAs using green fluorescent protein (GFP)-tagged FAK and FRNK. We report that Ca(2+) spikes are temporally correlated with movement and disassembly of FAs, but not their formation. FRNK transfection did not affect generation of Ca(2+) spikes, although cell morphology was altered, with fewer FAs of larger size and having a more peripheral localization being observed. Larger sized FAs in FRNK-transfected cells were not disassembled by Ca(2+) spikes, providing a possible explanation for impaired Ca(2+)-dependent migration in these cells. Stress fiber end movements initiated by Ca(2+) spikes were visualized using GFP-tagged myosin light chain kinase (MLCK). Ca(2+)-associated movements of stress fiber ends and FAs had similar kinetics, suggesting that stress fibers and FAs move in a coordinated fashion. This indicates that increases in Ca(2+) likely trigger disassembly of adhesive structures that involves disruption of integrin-extracellular matrix interactions, supporting a key role for Ca(2+)-sensitive inside-out signaling in cell migration. A rapid increase in tyrosine phosphorylation of FAK was found in response to an elevation in Ca(2+) induced by thapsigargin, and we propose that this represents the initial triggering event linking Ca(2+) signaling and FA dynamics to cell motility.

Rapid internalization and recycling of the human neuropeptide Y Y(1) receptor.

Desensitization of G protein-coupled receptors (GPCRs) involves receptor phosphorylation and reduction in the number of receptors at the cell surface. The neuropeptide Y (NPY) Y(1) receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y(1) receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y(1) receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y(1) receptors. Upon agonist challenge, the EGFP signal decreased rapidly (t(1/2) = 107 +/- 3 s) followed by a slow recovery. This decrease was blocked by BIBP3226, a Y(1) receptor antagonist, or by pertussis toxin, in agreement with Y(1) receptor activation. Internalization of EGFP-hNPY Y(1) receptors to acidic endosomal compartments likely accounts for the decrease in the EGFP signal, being absent after pretreatment with monensin. Concanavalin A and hypertonic sucrose, which inhibit clathrin-mediated endocytosis, blocked the decrease in fluorescence. After agonist, intracellular EGFP signals were punctate and co-localized with transferrin-Texas Red, a marker of clathrin-associated internalization and recycling, but not with LysoTracker Red, a lysosomal pathway marker, supporting receptor trafficking to recycling endosomes rather than the late endosomal/lysosomal pathway. Pulse-chase experiments revealed no receptor degradation after internalization. The slow recovery of fluorescence was unaffected by cycloheximide or actinomycin D, indicating that de novo synthesis of receptors was not limiting. Use of a multicompartment model to fit our fluorescence data allows simultaneous determination of internalization and recycling rate constants. We propose that rapid internalization of receptors via the clathrin-coated pits recycling pathway may largely account for the rapid desensitization of NPY Y(1) receptors.