Pharmacological and Genetic Disruption of C-Type Natriuretic Peptide (nppcl) Expression in Zebrafish (Danio rerio) Causes Stunted Growth during Development.

Human patients with mutations within NPPC or NPR2 genes (encoding C-type natriuretic peptide (CNP) and guanylyl cyclase-B (GC-B), respectively) display clinical signs associated with skeletal abnormalities, such as overgrowth or short stature. Mice with induced models of Nppc or Npr2 deletion display profound achondroplasia, dwarfism and early death. Recent pharmacological therapies to treat short stature are utilizing long-acting CNP analogues, but the effects of manipulating CNP expression during development remain unknown. Here, we use Danio rerio (zebrafish) as a model for vertebrate development, employing both pharmacological and reverse genetics approaches to alter expression of genes encoding CNP in zebrafish. Four orthologues of CNP were identified in zebrafish, and spatiotemporal expression profiling confirmed their presence during development. Bioinformatic analyses suggested that nppcl is the most likely the orthologue of mammalian CNP. Exogenous CNP treatment of developing zebrafish embryos resulted in impaired growth characteristics, such as body length, head width and eye diameter. This reduced growth was potentially caused by increased apoptosis following CNP treatment. Expression of endogenous nppcl was downregulated in these CNP-treated embryos, suggesting that negative feedback of the CNP system might influence growth during development. CRISPR knock-down of endogenous nppcl in developing zebrafish embryos also resulted in impaired growth characteristics. Collectively, these data suggest that CNP in zebrafish is crucial for normal embryonic development, specifically with regard to growth.

Mathematical models in GnRH research.

Mathematical modelling is an indispensable tool in modern biosciences, enabling quantitative analysis and integration of biological data, transparent formulation of our understanding of complex biological systems, and efficient experimental design based on model predictions. This review article provides an overview of the impact that mathematical models had on GnRH research. Indeed, over the last 20 years mathematical modelling has been used to describe and explore the physiology of the GnRH neuron, the mechanisms underlying GnRH pulsatile secretion, and GnRH signalling to the pituitary. Importantly, these models have contributed to GnRH research via novel hypotheses and predictions regarding the bursting behaviour of the GnRH neuron, the role of kisspeptin neurons in the emergence of pulsatile GnRH dynamics, and the decoding of GnRH signals by biochemical signalling networks. We envisage that with the advent of novel experimental technologies, mathematical modelling will have an even greater role to play in our endeavour to understand the complex spatiotemporal dynamics underlying the reproductive neuroendocrine system.

Modelling KNDy neurons and gonadotropin-releasing hormone pulse generation.

The pulsatile release of gonadotropin-releasing hormone (GnRH) and its frequency are crucial for healthy reproductive function. To understand what drives GnRH pulses, a combination of experimental and mathematical modelling approaches has been used. Early work focussed on the possibility that GnRH pulse generation is an intrinsic feature of GnRH neurons, with autocrine feedback generating pulsatility. However, there is now ample evidence suggesting that a network of upstream neurons secreting kisspeptin, neurokinin-B and dynorphin are the source of this GnRH pulse generator. The interplay of slow positive and negative feedback via neurokinin-B and dynorphin, respectively, allows the network to act as a relaxation oscillator, driving pulsatile secretion of kisspeptin, and consequently, of GnRH and LH. Here, we review the mathematical modelling approaches exploring both scenarios and suggest that with pulsatile GnRH secretion driven by the KNDy pulse generator, autocrine feedback still has the potential to modulate GnRH output.

IGFBP-1 expression is reduced in human type 2 diabetic glomeruli and modulates ß1-integrin/FAK signalling in human podocytes.

AIMS/HYPOTHESIS: Podocyte loss or injury is one of the earliest features observed in the pathogenesis of diabetic kidney disease (DKD), which is the leading cause of end-stage renal failure worldwide. Dysfunction in the IGF axis, including in IGF binding proteins (IGFBPs), is associated with DKD, particularly in the early stages of disease progression. The aim of this study was to investigate the potential roles of IGFBPs in the development of type 2 DKD, focusing on podocytes. METHODS: IGFBP expression was analysed in the Pima DKD cohort, alongside data from the Nephroseq database, and in ex vivo human glomeruli. Conditionally immortalised human podocytes and glomerular endothelial cells were studied in vitro, where IGFBP-1 expression was analysed using quantitative PCR and ELISAs. Cell responses to IGFBPs were investigated using migration, cell survival and adhesion assays; electrical cell-substrate impedance sensing; western blotting; and high-content automated imaging. RESULTS: Data from the Pima DKD cohort and from the Nephroseq database demonstrated a significant reduction in glomerular IGFBP-1 in the early stages of human type 2 DKD. In the glomerulus, IGFBP-1 was predominantly expressed in podocytes and controlled by phosphoinositide 3-kinase (PI3K)-forkhead box O1 (FoxO1) activity. In vitro, IGFBP-1 signalled to podocytes via ß1-integrins, resulting in increased phosphorylation of focal-adhesion kinase (FAK), increasing podocyte motility, adhesion, electrical resistance across the adhesive cell layer and cell viability. CONCLUSIONS/INTERPRETATION: This work identifies a novel role for IGFBP-1 in the regulation of podocyte function and that the glomerular expression of IGFBP-1 is reduced in the early stages of type 2 DKD, via reduced FoxO1 activity. Thus, we hypothesise that strategies to maintain glomerular IGFBP-1 levels may be beneficial in maintaining podocyte function early in DKD.

Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours.

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes.

Kisspeptin receptor agonist has therapeutic potential for female reproductive disorders.

BACKGROUNDKisspeptin is a key regulator of hypothalamic gonadotropin-releasing hormone (GnRH) neurons and is essential for reproductive health. A specific kisspeptin receptor (KISS1R) agonist could significantly expand the potential clinical utility of therapeutics targeting the kisspeptin pathway. Herein, we investigate the effects of a KISS1R agonist, MVT-602, in healthy women and in women with reproductive disorders.METHODSWe conducted in vivo and in vitro studies to characterize the action of MVT-602 in comparison with native kisspeptin-54 (KP54). We determined the pharmacokinetic and pharmacodynamic properties of MVT-602 (doses 0.01 and 0.03 nmol/kg) versus KP54 (9.6 nmol/kg) in the follicular phase of healthy women (n = 9), and in women with polycystic ovary syndrome (PCOS; n = 6) or hypothalamic amenorrhea (HA; n = 6). Further, we investigated their effects on KISS1R-mediated inositol monophosphate (IP1) and Ca2+ signaling in cell lines and on action potential firing of GnRH neurons in brain slices.RESULTSIn healthy women, the amplitude of luteinizing hormone (LH) rise was similar to that after KP54, but peaked later (21.4 vs. 4.7 hours; P = 0.0002), with correspondingly increased AUC of LH exposure (169.0 vs. 38.5 IU∙h/L; P = 0.0058). LH increases following MVT-602 were similar in PCOS and healthy women, but advanced in HA (P = 0.004). In keeping with the clinical data, MVT-602 induced more potent signaling of KISS1R-mediated IP1 accumulation and a longer duration of GnRH neuron firing than KP54 (115 vs. 55 minutes; P = 0.0012).CONCLUSIONTaken together, these clinical and mechanistic data identify MVT-602 as having considerable therapeutic potential for the treatment of female reproductive disorders.TRIAL REGISTRATIONInternational Standard Randomised Controlled Trial Number (ISRCTN) Registry, ISRCTN21681316.FUNDINGNational Institute for Health Research and NIH.

An information theoretic approach to insulin sensing by human kidney podocytes.

Podocytes are key components of the glomerular filtration barrier (GFB). They are insulin-responsive but can become insulin-resistant, causing features of the leading global cause of kidney failure, diabetic nephropathy. Insulin acts via insulin receptors to control activities fundamental to GFB integrity, but the amount of information transferred is unknown. Here we measure this in human podocytes, using information theory-derived statistics that take into account cell-cell variability. High content imaging was used to measure insulin effects on Akt, FOXO and ERK. Mutual Information (MI) and Channel Capacity (CC) were calculated as measures of information transfer. We find that insulin acts via noisy communication channels with more information flow to Akt than to ERK. Information flow estimates were increased by consideration of joint sensing (ERK and Akt) and response trajectory (live cell imaging of FOXO1-clover translocation). Nevertheless, MI values were always <1Bit as most information was lost through signaling. Constitutive PI3K activity is a predominant feature of the system that restricts the proportion of CC engaged by insulin. Negative feedback from Akt supressed this activity and thereby improved insulin sensing, whereas sensing was robust to manipulation of feedforward signaling by inhibiting PI3K, PTEN or PTP1B. The decisions made by individual podocytes dictate GFB integrity, so we suggest that understanding the information on which the decisions are based will improve understanding of diabetic kidney disease and its treatment.

A role for NPY-NPY2R signaling in albuminuric kidney disease.

Albuminuria is an independent risk factor for the progression to end-stage kidney failure, cardiovascular morbidity, and premature death. As such, discovering signaling pathways that modulate albuminuria is desirable. Here, we studied the transcriptomes of podocytes, key cells in the prevention of albuminuria, under diabetic conditions. We found that Neuropeptide Y (NPY) was significantly down-regulated in insulin-resistant vs. insulin-sensitive mouse podocytes and in human glomeruli of patients with early and late-stage diabetic nephropathy, as well as other nondiabetic glomerular diseases. This contrasts with the increased plasma and urinary levels of NPY that are observed in such conditions. Studying NPY-knockout mice, we found that NPY deficiency in vivo surprisingly reduced the level of albuminuria and podocyte injury in models of both diabetic and nondiabetic kidney disease. In vitro, podocyte NPY signaling occurred via the NPY2 receptor (NPY2R), stimulating PI3K, MAPK, and NFAT activation. Additional unbiased proteomic analysis revealed that glomerular NPY-NPY2R signaling predicted nephrotoxicity, modulated RNA processing, and inhibited cell migration. Furthermore, pharmacologically inhibiting the NPY2R in vivo significantly reduced albuminuria in adriamycin-treated glomerulosclerotic mice. Our findings suggest a pathogenic role of excessive NPY-NPY2R signaling in the glomerulus and that inhibiting NPY-NPY2R signaling in albuminuric kidney disease has therapeutic potential.

Regulation and Function of C-Type Natriuretic Peptide (CNP) in Gonadotrope-Derived Cell Lines.

C-type natriuretic peptide (CNP) is the most conserved member of the mammalian natriuretic peptide family, and is implicated in the endocrine regulation of growth, metabolism and reproduction. CNP is expressed throughout the body, but is particularly abundant in the central nervous system and anterior pituitary gland. Pituitary gonadotropes are regulated by pulsatile release of gonadotropin releasing hormone (GnRH) from the hypothalamus, to control reproductive function. GnRH and CNP reciprocally regulate their respective signalling pathways in αT3-1 gonadotrope cells, but effects of pulsatile GnRH stimulation on CNP expression has not been explored. Here, we examine the sensitivity of the natriuretic peptide system in LßT2 and αT3-1 gonadotrope cell lines to continuous and pulsatile GnRH stimulation, and investigate putative CNP target genes in gonadotropes. Multiplex RT-qPCR assays confirmed that primary mouse pituitary tissue express Nppc,Npr2 (encoding CNP and guanylyl cyclase B (GC-B), respectively) and Furin (a CNP processing enzyme), but failed to express transcripts for Nppa or Nppb (encoding ANP and BNP, respectively). Pulsatile, but not continuous, GnRH stimulation of LßT2 cells caused significant increases in Nppc and Npr2 expression within 4 h, but failed to alter natriuretic peptide gene expression in αT3-1 cells. CNP enhanced expression of cJun, Egr1, Nr5a1 and Nr0b1, within 8 h in LßT2 cells, but inhibited Nr5a1 expression in αT3-1 cells. Collectively, these data show the gonadotrope natriuretic peptide system is sensitive to pulsatile GnRH signalling, and gonadotrope transcription factors are putative CNP-target genes. Such findings represent additional mechanisms by which CNP may regulate reproductive function.

Determining the Relationship Between Hot Flushes and LH Pulses in Menopausal Women Using Mathematical Modeling.

BACKGROUND: Hypothalamic kisspeptin/neurokinin B/dynorphin (KNDy) neurones regulate LH pulsatility. It is widely accepted that the menopausal hot flush (HF) consistently synchronizes with the LH pulse, implicating the hypothalamic KNDy neurones in generating LH pulsatility and HF. Using a modern immunoassay and mathematical modeling, we investigated if the HF and LH pulse were consistently synchronized in menopausal women. METHODS: Eleven menopausal women (51 to 62 years of age and experiencing ≥7 HF in 24 hours) participated in an 8-hour study. Subjects self-reported HF and underwent peripheral blood sampling every 10 minutes. LH pulsatility was determined using two mathematical models: blinded deconvolution analysis and Bayesian spectrum analysis. The probability that the LH pulse and HF event intervals matched was estimated using the interval distributions observed in our data. RESULTS: Ninety-six HFs were self-reported, and 82 LH pulses were identified by blinded deconvolution analysis. Using both models, the probability that the two event intervals matched was low in the majority of participants (mean P = 0.24; P = 1 reflects perfect association). INTERPRETATION: Our data challenge the widely accepted dogma that HFs consistently synchronize with an LH pulse and therefore have clinically important therapeutic and mechanistic implications.

Measuring luteinising hormone pulsatility with a robotic aptamer-enabled electrochemical reader.

Normal reproductive functioning is critically dependent on pulsatile secretion of luteinising hormone (LH). Assessment of LH pulsatility is important for the clinical diagnosis of reproductive disorders, but current methods are hampered by frequent blood sampling coupled to expensive serial immunochemical analysis. Here, we report the development and application of a Robotic APTamer-enabled Electrochemical Reader (RAPTER) electrochemical analysis system to determine LH pulsatility. Through selective evolution of ligands by exponential enrichment (SELEX), we identify DNA aptamers that bind specifically to LH and not to related hormones. The aptamers are integrated into electrochemical aptamer-based (E-AB) sensors on a robotic platform. E-AB enables rapid, sensitive and repeatable determination of LH concentration profiles. Bayesian Spectrum Analysis is applied to determine LH pulsatility in three distinct patient cohorts. This technology has the potential to transform the clinical care of patients with reproductive disorders and could be developed to allow real-time in vivo hormone monitoring.

Exploring Dynamics and Noise in Gonadotropin-Releasing Hormone (GnRH) Signaling.

Gonadotropin-releasing hormone (GnRH) acts via G-protein coupled receptors on pituitary gonadotropes. These are Gq-coupled receptors that mediate acute effects of GnRH on the exocytotic secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as the chronic regulation of their synthesis. FSH and LH control steroidogenesis and gametogenesis in the gonads so GnRH mediates control of reproduction by the central nervous system. GnRH is secreted in short pulses and the effects of GnRH on its target cells are dependent on the dynamics of these pulses. Here we provide a brief overview of the signaling network activated by GnRH with emphasis on the use of high content imaging for their examination. We also describe computational approaches that we have used to simulate GnRH signaling in order to explore dynamics, noise, and information transfer in this system.

Enhanced insulin receptor, but not PI3K, signalling protects podocytes from ER stress.

Disruption of the insulin-PI3K-Akt signalling pathway in kidney podocytes causes endoplasmic reticulum (ER) stress, leading to podocyte apoptosis and proteinuria in diabetic nephropathy. We hypothesised that by improving insulin sensitivity we could protect podocytes from ER stress. Here we use established activating transcription factor 6 (ATF6)- and ER stress element (ERSE)-luciferase assays alongside a novel high throughput imaging-based C/EBP homologous protein (CHOP) assay to examine three models of improved insulin sensitivity. We find that by improving insulin sensitivity at the level of the insulin receptor (IR), either by IR over-expression or by knocking down the negative regulator of IR activity, protein tyrosine-phosphatase 1B (PTP1B), podocytes are protected from ER stress caused by fatty acids or diabetic media containing high glucose, high insulin and inflammatory cytokines TNFα and IL-6. However, contrary to this, knockdown of the negative regulator of PI3K-Akt signalling, phosphatase and tensin homolog deleted from chromosome 10 (PTEN), sensitizes podocytes to ER stress and apoptosis, despite increasing Akt phosphorylation. This indicates that protection from ER stress is conferred through not just the PI3K-Akt pathway, and indeed we find that inhibiting the MEK/ERK signalling pathway rescues PTEN knockdown podocytes from ER stress.

Gonadotropin-releasing hormone signaling: An information theoretic approach.

Gonadotropin-releasing hormone (GnRH) is a peptide hormone that mediates central control of reproduction, acting via G-protein coupled receptors that are primarily Gq coupled and mediate GnRH effects on the synthesis and secretion of luteinizing hormone and follicle-stimulating hormone. A great deal is known about the GnRH receptor signaling network but GnRH is secreted in short pulses and much less is known about how gonadotropes decode this pulsatile signal. Similarly, single cell measures reveal considerable cell-cell heterogeneity in responses to GnRH but the impact of this variability on signaling is largely unknown. Ordinary differential equation-based mathematical models have been used to explore the decoding of pulse dynamics and information theory-derived statistical measures are increasingly used to address the influence of cell-cell variability on the amount of information transferred by signaling pathways. Here, we describe both approaches for GnRH signaling, with emphasis on novel insights gained from the information theoretic approach and on the fundamental question of why GnRH is secreted in pulses.

Information Transfer via Gonadotropin-Releasing Hormone Receptors to ERK and NFAT: Sensing GnRH and Sensing Dynamics.

Information theoretic approaches can be used to quantify information transfer via cell signaling networks. In this study, we do so for gonadotropin-releasing hormone (GnRH) activation of extracellular signal-regulated kinase (ERK) and nuclear factor of activated T cells (NFAT) in large numbers of individual fixed LßT2 and HeLa cells. Information transfer, measured by mutual information between GnRH and ERK or NFAT, was <1 bit (despite 3-bit system inputs). It was increased by sensing both ERK and NFAT, but the increase was <50%. In live cells, information transfer via GnRH receptors to NFAT was also <1 bit and was increased by consideration of response trajectory, but the increase was <10%. GnRH secretion is pulsatile, so we explored information gained by sensing a second pulse, developing a model of GnRH signaling to NFAT with variability introduced by allowing effectors to fluctuate. Simulations revealed that when cell-cell variability reflects rapidly fluctuating effector levels, additional information is gained by sensing two GnRH pulses, but where it is due to slowly fluctuating effectors, responses in one pulse are predictive of those in another, so little information is gained from sensing both. Wet laboratory experiments revealed that the latter scenario holds true for GnRH signaling; within the timescale of our experiments (1 to 2 hours), cell-cell variability in the NFAT pathway remains relatively constant, so trajectories are reproducible from pulse to pulse. Accordingly, joint sensing, sensing of response trajectories, and sensing of repeated pulses can all increase information transfer via GnRH receptors, but in each case the increase is small.

Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes.

The natriuretic peptides, Atrial-, B-type and C-type natriuretric peptides (ANP, BNP, CNP), are regulators of many endocrine tissues and exert their effects predominantly through the activation of their specific guanylyl cyclase receptors (GC-A and GC-B) to generate cGMP. Whereas cGMP-independent signalling has been reported in response to natriuretic peptides, this is mediated via either the clearance receptor (Npr-C) or a renal-specific NPR-Bi isoform, which both lack intrinsic guanylyl cyclase activity. Here, we report evidence of GC-B-dependent cGMP-independent signalling in pituitary GH3 cells. Stimulation of GH3 cells with CNP resulted in a rapid and sustained enhancement of ERK1/2 phosphorylation (P-ERK1/2), an effect that was not mimicked by dibutryl-cGMP. Furthermore, CNP-stimulated P-ERK1/2 occurred at concentrations below that required for cGMP accumulation. The effect of CNP on P-ERK1/2 was sensitive to pharmacological blockade of MEK (U0126) and Src kinases (PP2). Silencing of the GC-B1 and GC-B2 splice variants of the GC-B receptor by using targeted short interfering RNAs completely blocked the CNP effects on P-ERK1/2. CNP failed to alter GH3 cell proliferation or cell cycle distribution but caused a concentration-dependent increase in the activity of the human glycoprotein α-subunit promoter (αGSU) in a MEK-dependent manner. Finally, CNP also activated the p38 and JNK MAPK pathways in GH3 cells. These findings reveal an additional mechanism of GC-B signalling and suggest additional biological roles for CNP in its target tissues.

Targeted disruption of the orphan receptor Gpr151 does not alter pain-related behaviour despite a strong induction in dorsal root ganglion expression in a model of neuropathic pain.

BACKGROUND: Gpr151 is an orphan GPCR whose function is unknown. The restricted pattern of neuronal expression in the habenula, dorsal horn of the spinal cord and dorsal root ganglion plus homology with the galanin family of receptors imply a role in nociception. RESULTS: Real-time quantitative RT-PCR demonstrated a 49.9±2.9 fold highly significant (P<0.001) increase in Gpr151 mRNA expression in the dorsal root ganglion 7days after the spared nerve injury model of neuropathic pain. Measures of acute, inflammatory and neuropathic pain behaviours were not significantly different using separate groups of Gpr151 loss-of-function mutant mice and wild-type controls. Galanin at concentrations between 100nM and 10µM did not induce calcium signalling responses in ND7/23 cells transfected with Gpr151. CONCLUSIONS: Our results indicate that despite the very large upregulation in the DRG after a nerve injury model of neuropathic pain, the Gpr151 orphan receptor does not appear to be involved in the modulation of pain-related behaviours. Further, galanin is unlikely to be an endogenous ligand for Gpr151.

Mathematical modeling of gonadotropin-releasing hormone signaling.

Gonadotropin-releasing hormone (GnRH) acts via G-protein coupled receptors on pituitary gonadotropes to control reproduction. These are Gq-coupled receptors that mediate acute effects of GnRH on the exocytotic secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), as well as the chronic regulation of their synthesis. GnRH is secreted in short pulses and GnRH effects on its target cells are dependent upon the dynamics of these pulses. Here we overview GnRH receptors and their signaling network, placing emphasis on pulsatile signaling, and how mechanistic mathematical models and an information theoretic approach have helped further this field.

Agonist-induced internalization and desensitization of the apelin receptor.

Apelin acts via the G protein-coupled apelin receptor (APJ) to mediate effects on cardiovascular and fluid homeostasis. G protein-coupled receptor (GPCR) trafficking has an important role in the regulation of receptor signalling pathways and cellular functions, however in the case of APJ the mechanisms and proteins involved in apelin-induced trafficking are not well understood. We generated a stable HEK-293 cell line expressing N-terminus HA-tagged mouse (m) APJ, and used a semi-automated imaging protocol to quantitate APJ trafficking and ERK1/2 activation following stimulation with [Pyr1]apelin-13. The mechanisms of [Pyr1]apelin-13-induced internalization and desensitization were explored using dominant-negative mutant (DNM) cDNA constructs of G protein-coupled receptor kinase 2 (GRK2), ß-arrestin1, EPS15 and dynamin. The di-phosphorylated ERK1/2 (ppERK1/2) response to [Pyr1]apelin-13 desensitized during sustained stimulation, due to upstream APJ-specific adaptive changes. Furthermore, [Pyr1]apelin-13 stimulation caused internalization of mAPJ via clathrin coated vesicles (CCVs) and also caused a rapid reduction in cell surface and whole cell HA-mAPJ. Our data suggest that upon continuous agonist exposure GRK2-mediated phosphorylation targets APJ to CCVs that are internalized from the cell surface in a ß-arrestin1-independent, EPS15- and dynamin-dependent manner. Internalization does not appear to contribute to the desensitization of APJ-mediated ppERK1/2 activation in these cells.