Determination of EGFR Endocytosis Kinetic by Auto-Regulatory Association of PLD1 with μ2

Background

Upon ligand binding, cell surface signaling receptors are internalized through a process tightly regulated by endocytic proteins and adaptor protein 2 (AP2) to orchestrate them. Although the molecular identities and roles of endocytic proteins are becoming clearer, it is still unclear what determines the receptor endocytosis kinetics which is mainly regulated by the accumulation of endocytic apparatus to the activated receptors.

Methodology/Principal Findings

Here we employed the kinetic analysis of endocytosis and adaptor recruitment to show that μ2, a subunit of AP2 interacts directly with phospholipase D (PLD)1, a receptor-associated signaling protein and this facilitates the membrane recruitment of AP2 and the endocytosis of epidermal growth factor receptor (EGFR). We also demonstrate that the PLD1-μ2 interaction requires the binding of PLD1 with phosphatidic acid, its own product.

Conclusions/Significance

These results suggest that the temporal regulation of EGFR endocytosis is achieved by auto-regulatory PLD1 which senses the receptor activation and triggers the translocation of AP2 near to the activated receptor.     

Differential Sensitivity of the Short and Long Human Dopamine D2 Receptor Subtypes to Protein Kinase C

The human dopamine D2L (long form) and D2S (short form) receptors were expressed separately in mouse Ltk- fibroblast cells to investigate whether there is a difference in transmembrane signaling of these D2 receptors. Both receptors induced two signals, a phosphatidylinositol-linked mobilization of intracellular calcium and an inhibition of cyclic adenosine 3'-5' monophosphate (cAMP) accumulation, each with similar response magnitudes and identical pharmacology. Both calcium and cAMP signals were sensitive to pretreatment with pertussis toxin (PTX), indicating mediation by coupling to Gi/Go proteins. However, the two forms of D2 receptor were distinguished by acute prior activation of protein kinase C (PKC) with 12-O-tetradecanoyl 4 beta-phorbol 13-acetate (TPA): TPA blocked the D2S-mediated increase in cytosolic free calcium concentration ([Ca2+]i) in a concentration-dependent manner (between 10 nM and 1 microM), whereas the D2L receptor-induced increase in [Ca2+]i was resistant to TPA and was only partially (60%) inhibited by 100 microM TPA. By contrast, TPA did not alter the inhibition of cAMP accumulation induced by activation of either D2S or D2L receptors. We conclude that, in the L cell system, prior activation of PKC differentially modulates the transmembrane signaling of the D2L and D2S receptors, preferentially inhibiting the D2S receptor-mediated calcium signal but not altering the dopamine-induced inhibitory cAMP signal of either receptor subtype.     

Opposing effects of phorbol-12-myristate-13-acetate, an activator of protein kinase C, on the signaling of structurally related human dopamine D1 and D5 receptors

The ‘cross‐talk’ between different types of neurotransmitters through second messenger pathways represents a major regulatory mechanism in neuronal function. We investigated the effects of activation of protein kinase C (PKC) on cAMP‐dependent signaling by structurally related human D1‐like dopaminergic receptors. Human embryonic kidney 293 (HEK293) cells expressing D1 or D5 receptors were pretreated with phorbol‐12‐myristate‐13‐acetate (PMA), a potent activator of PKC, followed by analysis of dopamine‐mediated receptor activation using whole cell cAMP assays. Unpredictably, PKC activation had completely opposite effects on D1 and D5 receptor signaling. PMA dramatically augmented agonist‐evoked D1 receptor signaling, whereas constitutive and dopamine‐mediated D5 receptor activation were rapidly blunted. RT–PCR and immunoblotting analyses showed that phorbol ester‐regulated PKC isozymes (conventional: α, βI, βII, γ; novel: δ, ?, η, θ) and protein kinase D (PKCµ) are expressed in HEK293 cells. PMA appears to mediate these contrasting effects through the activation of Ca²⁺‐independent novel PKC isoforms as revealed by specific inhibitors, bisindolylmaleimide I, Gö6976, and Gö6983. The finding that cross‐talk between PKC and cAMP pathways can produce such opposite outcomes following the activation of structurally similar D1‐like receptor subtypes is novel and further strengthens the view that D1 and D5 receptors serve distinct functions in the mammalian nervous and endocrine systems.     

Autocrine activation of adenosine A1 receptors blocks D1A but not D1B dopamine receptor desensitization

Adenosine is known to modulate dopamine responses in several brain areas. Here, we show that tonic activation of adenosine receptors is able to impede desensitization of D1 dopamine receptors. As measured by cAMP accumulation in transfected COS-7 cells, long-term exposure to dopamine agonists promoted desensitization of D1B receptor but not that of D1A receptor. The inability of D1A receptor to desensitize was a result of the adenosine present in culture medium acting through activation of adenosine A1 receptors. Cell incubation with either adenosine deaminase, CGS-15943, a generic adenosine receptor antagonist, or the A1 antagonist DPCPX restored the long-term desensitization time-course of D1A receptors. In Ltk cells stably expressing A1 adenosine receptors and D1A dopamine receptors, pre-treatment of cells with R(-)-PIA, a full A1 receptor agonist, did not significantly inhibit the acute increase in cAMP levels induced by D1 receptor agonists, but blocked desensitization of D1A receptors. However, simultaneous activation of A1 and D1A receptors promoted a delayed D1A receptor desensitization. This suggests that functional interaction between A1 and D1A receptors may depend on the activation kinetics of components regulating D1 receptor responses, acting differentially on D1A and D1B receptors.     

α1A-adrenergic receptor induces activation of extracellular signal-regulated kinase 1/2 through endocytic pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α(1A)-adrenergic receptor (α(1A)-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α(1A)-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α(1A)-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α(1A)-AR. α(1A)-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α(1A)-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31-8220 (a PKC inhibitor) inhibited α(1B)-AR- but not α(1A)-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α(1A)-AR-induced ERK1/2 activation, which is independent of G(q)/PLC/PKC signaling.     

Differential regulation of dopamine D1 and D2 signaling by nicotine in neostriatal neurons

Nicotine, acting on nicotinic acetylcholine receptors (nAChRs) expressed at pre-synaptic dopaminergic terminals, has been shown to stimulate the release of dopamine in the neostriatum. However, the molecular consequences of pre-synaptic nAChR activation in post-synaptic neostriatal neurons are not clearly understood. Here, we investigated the effect of nAChR activation on dopaminergic signaling in medium spiny neurons by measuring phosphorylated DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa) at Thr34 (the PKA-site) in mouse neostriatal slices. Nicotine produced dose-dependent responses, with a low concentration (1 microm) causing a sustained decrease in DARPP-32 Thr34 phosphorylation and a high concentration (100 microm) causing a transient increase in DARPP-32 Thr34 phosphorylation. Depending on the concentration of nicotine, either dopamine D2 or D1 receptor signaling was predominantly activated. Nicotine at a low concentration (1 microm) activated dopamine D2 receptor signaling in striatopallidal/indirect pathway neurons, likely by activating alpha4beta2* nAChRs at dopaminergic terminals. Nicotine at a high concentration (100 microm) activated dopamine D1 receptor signaling in striatonigral/direct pathway neurons, likely by activating (i) alpha4beta2* nAChRs at dopaminergic terminals and (ii) alpha7 nAChRs at glutamatergic terminals, which, by stimulating the release of glutamate, activated NMDA/AMPA receptors at dopaminergic terminals. The differential effects of low and high nicotine concentrations on D2- and D1-dependent signaling pathways in striatal neurons may contribute to dose-dependent actions of this drug of abuse.     

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

Endocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wild-type Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and endocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.     

Inhibition of the dopamine D1 receptor signaling by PSD-95

Dopamine D1 receptors play an important role in movement, reward, and learning and are implicated in a number of neurological and psychiatric disorders. These receptors are concentrated in dendritic spines of neurons, including the spine head and the postsynaptic density. D1 within spines is thought to modulate the local channels and receptors to control the excitability and synaptic properties of spines. The molecular mechanisms mediating D1 trafficking, anchorage, and function in spines remain elusive. Here we show that the synaptic scaffolding protein PSD-95 thought to play a role in stabilizing glutamate receptors in the postsynaptic density, interacts with D1 and regulates its trafficking and function. Interestingly, the D1-PSD-95 interaction does not require the well characterized domains of PSD-95 but is mediated by the carboxyl-terminal tail of D1 and the NH(2) terminus of PSD-95, a region that is recognized only recently to participate in protein-protein interaction. Co-expression of PSD-95 with D1 in mammalian cells inhibits the D1-mediated cAMP accumulation without altering the total expression level or the agonist binding properties of the receptor. The diminished D1 signaling is mediated by reduced D1 expression at the cell surface as a consequence of an enhanced constitutive, dynamin-dependent endocytosis. In addition, genetically engineered mice lacking PSD-95 show a heightened behavioral response to either a D1 agonist or the psychostimulant amphetamine. These studies demonstrate a role for a glutamatergic scaffold in dopamine receptor signaling and trafficking and identify a new potential target for the modulation of abnormal dopaminergic function.     

Signaling on the endocytic pathway

Ligand binding to receptor tyrosine kinases and G-protein-coupled receptors initiates signal transduction events and induces receptor endocytosis via clathrin-coated pits and vesicles. While receptor-mediated endocytosis has been traditionally considered an effective mechanism to attenuate ligand-activated responses, more recent studies demonstrate that signaling continues on the endocytic pathway. In fact, certain signaling events, such as the activation of the extracellular signal-regulated kinases, appear to require endocytosis. Protein components of signal transduction cascades can assemble at clathrin coated pits and remain associated with endocytic vesicles following their dynamin-dependent release from the plasma membrane. Thus, endocytic vesicles can function as a signaling compartment distinct from the plasma membrane. These observations demonstrate that endocytosis plays an important role in the activation and propagation of signaling pathways.     

Parathyroid hormone receptor trafficking contributes to the activation of extracellular signal-regulated kinases but is not required for regulation of cAMP signaling

Agonist-mediated activation of the type 1 parathyroid hormone receptor (PTH1R) results in several signaling events and receptor endocytosis. It is well documented that arrestins contribute to desensitization of both G(s)- and G(q)-mediated signaling and mediate PTH1R internalization. However, whether PTH1R trafficking directly contributes to signaling remains unclear. To address this question, we investigated the role of PTH1R trafficking in cAMP signaling and activation of extracellular signal-regulated kinases ERK1/2 in HEK-293 cells. Dominant negative forms of dynamin (K44A-dynamin) and beta-arrestin1 (beta-arrestin1-(319-418)) abrogated PTH1R internalization but had no effect on cAMP signaling; neither acute cAMP production by PTH nor desensitization and resensitization of cAMP signaling were affected. Therefore, PTH1R trafficking is not necessary for regulation of cAMP signaling. PTH-(1-34) induced rapid and robust activation of ERK1/2. A PTHrP-based analog ([p-benzoylphenylalanine1, Ile5,Arg(11,13),Tyr36]PTHrP-(1-36)NH2), which selectively activates the G(s)/cAMP pathway without inducing PTH1R endocytosis, failed to stimulate ERK1/2 activity. Inhibition of PTH1R endocytosis by K44A-dynamin dampened ERK1/2 activation in response to PTH-(1-34) by 69%. Incubation with the epidermal growth factor receptor inhibitor AG1478 reduced ERK1/2 phosphorylation further. In addition, ERK1/2 phosphorylation occurred following internalization of a PTH1R mutant induced by PTH-(7-34) in the absence of G protein signaling. Collectively, these data indicate that PTH1R trafficking and G(q) (but not G(s)) signaling independently contribute to ERK1/2 activation, predominantly via transactivation of the epidermal growth factor receptor.     

Endocytic downregulation of ErbB receptors: mechanisms and relevance in cancer

ErbB receptors (EGFR (ErbB1), ErbB2, ErbB3, and ErbB4) are important regulators of normal growth and differentiation, and they are involved in the pathogenesis of cancer. Following ligand binding and receptor activation, EGFR is endocytosed and transported to lysosomes where the receptor is degraded. This downregulation of EGFR is a complex and tightly regulated process. The functions of ErbB2, ErbB3, and ErbB4 are also regulated by endocytosis to some extent, although the current knowledge of these processes is sparse. Impaired endocytic downregulation of signaling receptors is frequently associated with cancer, since it can lead to increased and uncontrolled receptor signaling. In this review we describe the current knowledge of ErbB receptor endocytic downregulation. In addition, we outline how ErbB receptors can escape endocytic downregulation in cancer, and we discuss how targeted anti-cancer therapy may induce endocytic downregulation of ErbB receptors.     

Epidermal growth factor and membrane trafficking. EGF receptor activation of endocytosis requires Rab5a

Activated epidermal growth factor receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between signaling and endocytosis is not well understood. Here we show that EGF stimulation of NR6 cells induces a specific, rapid and transient activation of Rab5a. EGF also enhanced translocation of the Rab5 effector, early endosomal autoantigen 1 (EEA1), from cytosol to membrane. The activation of endocytosis, fluid phase and receptor mediated, by EGF was enhanced by Rab5a expression, but not by Rab5b, Rab5c, or Rab5a truncated at the NH(2) and/or COOH terminus. Dominant negative Rab5a (Rab5:N34) blocked EGF-stimulated receptor-mediated and fluid-phase endocytosis. EGF activation of Rab5a function was dependent on tyrosine residues in the COOH-terminal domain of the EGF receptor (EGFR). Removal of the entire COOH terminus by truncation (c'973 and c'991) abrogated ligand-induced Rab5a activation of endocytosis. A "kinase-dead" EGFR failed to stimulate Rab5a function. However, another EGF receptor mutant (c'1000), with the kinase domain intact and a single autophosphorylation site effectively signaled Rab5 activation. These results indicate that EGFR and Rab5a are linked via a cascade that results in the activation of Rab5a and that appears essential for internalization. The results point to an interdependent relationship between receptor activation, signal generation and endocytosis.     

Trafficking properties of the D5 dopamine receptor

Dopamine receptors are important for diverse biological functions and are important pharmacological targets in human medicine. Signal transduction from the dopamine receptors is controlled at many levels, including by the process of receptor trafficking. Little is known regarding the endocytic and postendocytic trafficking properties of the D5 dopamine receptor. Here, we show that endocytosis of the D5 receptor can be achieved both homologously, through direct receptor activation by agonist, and also heterologously, due to independent activation of protein kinase C (PKC). In contrast, the D1 receptor is endocytosed only in response to agonist but not PKC activation. We have identified the residue in the third intracellular loop of the D5 receptor that is both necessary for PKC-mediated endocytosis of the D5 receptor and sufficient to induce PKC-mediated endocytosis when introduced to the D1 receptor. In addition, we show that endocytosis of D5 through both pathways is dependent on clathrin and dynamin but that only agonist-induced endocytosis engages β-arrestin 2. Together, these data show that the D5 receptor shows a trafficking profile distinct from that of any of the other dopamine receptors.     

Homologous regulation of the heptahelical D1A receptor responsiveness: specific cytoplasmic tail regions mediate dopamine-induced phosphorylation,desensitization and endocytosis

In the present study, we investigate the role of specific cytoplasmic tail (CT) regions of the D1A receptor in mediating dopamine (DA)-induced phosphorylation, desensitization and endocytosis. Results obtained in human embryonic kidney (HEK) cells expressing the wild-type (WT) or truncation forms (Delta425, Delta379 and Delta351) of the D1A receptor show that sequences located downstream of Gly379 regulate DA-mediated phosphorylation-dependent desensitization of D1A receptors. However, the longer truncation mutant Delta351 failed to undergo detectable DA-induced phosphorylation while exhibiting DA-induced desensitization features similar to the shorter truncation mutant Delta379. These data potentially suggest a novel role for a receptor phosphorylation-independent process in the DA-promoted D1A subtype desensitization. Our immunofluorescence data also suggest that sequences located between Cys351 and Gly379 play an important role in DA-mediated receptor endocytosis. Additionally, time-course studies were done in intact cells expressing WT or truncation receptors to measure the observed rate constant for adenylyl cyclase (AC) activation or k(obs), a parameter linked to the receptor-G protein coupling status. In agreement with the desensitization data, Delta425- and Delta379-expressing cells exhibit an increase of kobs in comparison with WT-expressing cells. Nevertheless, Delta351-expressing cells, which harbor similar desensitization features of Delta379-expressing cells, display no change in k(obs) when compared with WT-expressing cells. Our results suggest that a defective DA-induced endocytosis may hamper Delta351 resensitization and concomitant increase in k(obs). Thus, our study showing that specific D1A receptor CT sequences regulate DA-induced phosphorylation, desensitization, and endocytosis highlights the underlying molecular complexity of signaling at dopaminergic synapses.     

c-Src is involved in regulating signal transmission from PDGFbeta receptor-GPCR(s) complexes in mammalian cells

We have reported that the platelet-derived growth factor receptor-beta (PDGFbeta) forms a novel signaling complex with G protein-coupled receptors (GPCR) (e.g. S1P(1) receptor) that enables more efficient activation of p42/p44 mitogen-activated protein kinase (MAPK) in response to PDGF and sphingosine 1-phosphate (S1P). We now demonstrate that c-Src participates in regulating the endocytosis of PDGFbeta receptor-GPCR complexes in response to PDGF. This leads to association of cytoplasmic p42/p44 MAPK with the receptor complex in endocytic vesicles. c-Src is regulated by G protein betagamma subunits and can interact with beta-arrestin. Indeed, the PDGF-dependent activation of p42/p44 MAPK was reduced by over-expression of the C-terminal domain of GRK2 (sequesters Gbetagamma subunits), the clathrin-binding domain of beta-arrestin and by inhibitors of c-Src and clathrin-mediated endocytosis. Moreover, PDGF and S1P induce the recruitment of c-Src to the PDGFbeta receptor-S1P(1) receptor complex. This leads to a G protein/c-Src-dependent tyrosine phosphorylation of Gab1 and accumulation of dynamin II at the plasma membrane, a step required for endocytosis of the PDGFbeta receptor-GPCR complex. These findings provide important information concerning the molecular organisation of novel receptor tyrosine kinase (RTK)-GPCR signal relays in mammalian cells.     

Dopamine-dependent ectodomain shedding and release of epidermal growth factor in developing striatum: target-derived neurotrophic signaling (Part 2)

Epidermal growth factor (EGF) and structurally related peptides promote neuronal survival and the development of midbrain dopaminergic neurons; however, the regulation of their production has not been fully elucidated. In this study, we found that the treatment of striatal cells with dopamine agonists enhances EGF release both in vivo and in vitro. We prepared neuron-enriched and non-neuronal cell-enriched cultures from the striatum of rat embryos and challenged those with various neurotransmitters or dopamine receptor agonists. Dopamine and a dopamine D(1) -like receptor agonist (SKF38393) triggered EGF release from neuron-enriched cultures in a dose-dependent manner. A D(2) -like agonist (quinpirole) increased EGF release only from non-neuronal cell-enriched cultures. The EGF release from striatal neurons and non-neuronal cells was concomitant with ErbB1 phosphorylation and/or with the activation of a disintegrin and metalloproteinase and matrix metalloproteinase. The EGF release from neurons was attenuated by an a disintegrin and metalloproteinase/matrix metalloproteinase inhibitor, GM6001, and a calcium ion chelator, BAPTA/AM. Transfection of cultured striatal neurons with alkaline phosphatase-tagged EGF precursor cDNA confirmed that dopamine D(1) -like receptor stimulation promoted both ectodomain shedding of the precursor and EGF release. Therefore, the activation of striatal dopamine receptors induces shedding and release of EGF to provide a retrograde neurotrophic signal to midbrain dopaminergic neurons.     

D1 dopamine receptor mediates dopamine-induced cytotoxicity via the ERK signal cascade

Postsynaptic striatal neurodegeneration occurs through unknown mechanisms, but it is linked to high extracellular levels of synaptic dopamine. Dopamine-mediated cytotoxicity of striatal neurons occurs through two distinct pathways: autoxidation and the D1 dopamine receptor-linked signaling pathway. Here we investigated the mitogen-activated protein kinase (MAPK) signaling pathways activated upon the acute stimulation of D1 dopamine receptors. In SK-N-MC neuroblastoma cells, endogenously expressing D1 dopamine receptors, dopamine caused activation of phosphorylated (p-)ERK1/2 and of the stress-signaling kinases, p-JNK and p-p38 MAPK, in a time- and dose-dependent manner. Selective stimulation of D1 receptors with the agonist SKF R-38393 caused p-ERK1/2, but not p-JNK or p-p38 MAPK activation, in a manner sensitive to the receptor-selective antagonist SCH 23390, protein kinase A inhibition (KT5720), and MEK1/2 inhibition (U0126 or PD98059). Activation of ERK by D1 dopamine receptors resulted in oxidative stress and cytotoxicity. In cells transfected with a catalytically defective mutant of MEK1, the upstream ERK-specific kinase, both dopamine- and SKF R-38393-mediated cytotoxicity was markedly attenuated, confirming the participation of the ERK signaling pathway. Cell fractionation studies showed that only a small amount of p-ERK1/2 was translocated to the nucleus, with the majority retained in the cytoplasm. From coimmunoprecipitation studies, p-ERK was found to form stable heterotrimeric complexes with the D1 dopamine receptor and beta-arrestin2. In cells transfected with the dominant negative mutant of beta-arrestin2, the formation of such complexes was substantially inhibited. These data provide novel mechanistic insights into the role of ERK in the cytotoxicity mediated upon activation of the D1 dopamine receptor.     

Kinin-Stimulated B1 Receptor Signaling Depends on Receptor Endocytosis Whereas B2 Receptor Signaling Does Not

Kinins are potent pro-inflammatory peptides that act through two G protein-coupled receptor subtypes, B1 (B1R) and B2 (B2R). Kinin-stimulated B2R signaling is often transient, whereas B1R signaling is sustained. This was confirmed by monitoring agonist-stimulated intracellular Ca²⁺ mobilization in A10 smooth muscle cells expressing human wild-type B2R and B1R. We further studied the role of receptor membrane trafficking in receptor-mediated phosphoinositide (PI) hydrolysis in model HEK293 cell lines stably expressing the receptors. Treatment of cells with brefeldin A, to inhibit maturation of de novo synthesized receptors, or hypertonic sucrose, to inhibit receptor endocytosis, showed that the basal cell surface receptor turnover was considerably faster for B1R than for B2R. Inhibition of endocytosis, which stabilized B1R on the cell surface, inhibited B1R signaling, whereas B2R signaling was not perturbed. Signaling by a B1R construct in which the entire C-terminal domain was deleted remained sensitive to inhibition of receptor endocytosis, whereas signaling by a B1R construct in which this domain was substituted with the corresponding domain in B2R was not sensitive. B2R and B1R co-expression, which also appeared to stabilize B1R on the cell surface, presumably by receptor hetero-dimerization, also inhibited B1R signaling, whereas B2R signaling was slightly enhanced. Furthermore, the B2R-specific agonist bradykinin (BK) directed both receptors through a common endocytic pathway, whereas the B1R-specific agonist Lys-desArg⁹-BK was unable to do so. These results suggest that B1R-mediated PI hydrolysis depends on a step in receptor endocytosis, whereas B2R-mediated PI hydrolysis does not. We propose that B1R uses at least part of the endocytic machinery to sustain agonist-promoted signaling.