Regulation of lateral mobility and cellular trafficking of the CCK receptor by a partial agonist

Partial agonists are effective tools for advancing developmentof highly selective drugs and providing insights into molecular regulation of cellular functions. Here, we explore the impact of apartial agonist on key aspects of cholecystokinin (CCK) receptor regulation, its lateral mobility and cellular trafficking, in nativepancreatic acinar cells and Chinese hamster ovary cells expressing CCKreceptor (CHO-CCKR). We developed and characterized a novel fluorescentpartial agonist,rhodamine-Gly-[(Nle^28,31)CCK-26-32]-phenethylester, that binds specifically and with high affinity to CCK receptors.Such analogs are fully efficacious pancreatic acinar cell secretagogueswithout supramaximal inhibition that mobilize intracellular calciumwith little or no increase in phospholipase C (PLC) activity. Despiteminimal phosphorylation of CCK receptors in response to this partialagonist, receptor trafficking was the same as that observed with fullagonist (CCK). This included normal internalization viaclathrin-dependent endocytosis in CHO-CCKR cells and insulation on thesurface of pancreatic acinar cells. Also, as with CCK-occupiedreceptor, fluorescence recovery after photobleaching of partialagonist-occupied receptor on the acinar cell surface demonstrated amarked temperature-dependent slowing of its rate of diffusion. This wassimilarly associated with resistance to acid-induced dissociation ofligand. Thus some key molecular regulatory mechanisms for CCK receptorinternalization and insulation may be initiated by cellular signalingcascades that are not dependent on PLC activation or receptor phosphorylation.     

Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell

Receptor desensitization is a key process for the protection of the cell from continuous or repeated exposure to high concentrations of an agonist. Well-established mechanisms for desensitization of guanine nucleotide-binding protein (G protein)-coupled receptors include phosphorylation, sequestration/internalization, and down-regulation. In this work, we have examined some mechanisms for desensitization of the cholecystokinin (CCK) receptor which is native to the pancreatic acinar cell, and have found the predominant mechanism to be distinct from these recognized processes. Upon fluorescent agonist occupancy of the native receptor, it becomes "insulated" from the effects of acid washing and becomes immobilized on the surface of the plasma membrane in a time- and temperature-dependent manner. This localization was assessed by ultrastructural studies using a colloidal gold conjugate of CCK, and lateral mobility of the receptor was assessed using fluorescence recovery after photobleaching. Of note, recent application of the same morphologic techniques to a CCK receptor-bearing Chinese hamster ovary cell line demonstrated prominent internalization via the clathrin-dependent endocytic pathway, as well as entry into caveolae (Roettger, B.F., R.U. Rentsch, D. Pinon, E. Holicky, E. Hadac, J.M. Larkin, and L.J. Miller, 1995, J. Cell Biol. 128: 1029-1041). These organelles are not observed to represent prominent compartments for the same receptor to traverse in the acinar cell, although fluorescent insulin is clearly internalized in these cells via receptor-mediated endocytosis. In this work, the rate of lateral mobility of the CCK receptor is observed to be similar in both cell types (1-3 x 10(-10) cm2/s), while the fate of the agonist-occupied receptor is quite distinct in each cell. This supports the unique nature of desensitization processes which occur in a cell-specific manner. A plasmalemmal site of insulation of this important receptor on the pancreatic acinar cell could be particularly effective to protect the cell from processes which might initiate pancreatitis, while providing for the rapid resensitization of this receptor to ensure appropriate pancreatic secretion to aid in nutrient assimilation for the organism.     

Signal Flows from Two Phospholipase C-Linked Receptors Are Independent in PC12 Cells

Abstract: Bradykinin (BK) receptor and P₂-purinergic receptor are known to be coupled to phospholipase C (PLC) in PC12 cells. To study the interaction between these two PLC-linked receptors, the presence of both receptors on individual cells was demonstrated by sequential Ca²⁺ spikes caused by BK and ATP in a single fura-2-loaded cell. BK- and ATP-induced catecholamine (CA) secretions were desensitized within 5 min. However, in the sequential experiment, the BK-induced homologous desensitization of CA secretion did not block the ATP-induced secretion, and vice versa. Each agonist-induced an increase in inositol 1,4,5-trisphosphate (IP₃) production and intracellular free Ca²⁺ concentration also led to homologous desensitization. However, there was no heterologous desensitization between the two agonists. When the cells were treated with both BK and ATP simultaneously, the amounts of CA secretion, IP₃ production, internal Ca²⁺ mobilization, and Ca²⁺ influx were all additive. We also found that both IP₃-induced Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ influx from extracellular space were able to release [³H]norepinephrine, and the secretion induced by both agonists was exactly additive in the absence or presence of extracellular Ca²⁺. The data suggest that the CA secretions caused by BK or ATP may have separate secretory pathways even though they activate identical second messenger pathways.     

Desensitization of the Neurokinin 1 Receptor Is Mediated by the Receptor Carboxy-Terminal Region, but Is Not Caused by Receptor Internalization

Abstract: The carboxy-terminal cytoplasmic regions of the rat neurokinin 1 (substance P) and neurokinin 2 (neurokinin A) receptors have been exchanged to determine if this region of the neurokinin 1 receptor is involved in its desensitization. When expressed at similar levels in stably transfected Chinese hamster ovary (CHO) cell lines, receptors containing the carboxy-terminal region of the neurokinin 1 receptor desensitized significantly more (as measured by reduction of the inositol 1,4,5-trisphosphate response) when preexposed for 1 min to 1 µ M neurokinin, indicating a role for the carboxy-terminal region of the neurokinin 1 receptor in its desensitization. Measurement of receptor internalization using radiolabeled neurokinins (0.3 n M ) indicated that ∼75–80% of the receptors were internalized in each cell line after 10 min at 37°C, with no observable correlation between neurokinin receptor desensitization and internalization. Measurement of loss of receptor surface sites for cell lines CHO NK1 and CHO NK1NK2 following exposure to 1 µ M substance P also indicated no obvious relationship between the percent desensitization and percent of receptors internalized. Also, two inhibitors of neurokinin 1 receptor internalization, phenylarsine oxide and hyperosmolar sucrose, did not inhibit neurokinin 1 receptor desensitization. The protein kinase inhibitors Ro 31-8220, staurosporine, and Zn²⁺ had no effect on neurokinin 1 receptor desensitization, indicating that the kinases affected by these agents are not rate-limiting in neurokinin 1 receptor desensitization in this system.     

Identification of the GABAA Receptor α3 Subunit in the IMR-32 Neuroblastoma Cell Line

Abstract: A previous report has described the presence of f-[³⁵S]- butylbicyclophosphorothionate binding sites and GABA-gated CI flux in the human neuroblastoma IMR-32 cell line. We now report the further characterisation of this binding site and, even more important, the identification of the GABA_A receptor α3 subunit expressed in these cells. Cell membranes prepared from IMR-32 cells were screened by immunoblotting for reactivity with various GABA_A receptor a subunit-specific antibodies. Of these, only anti-Cys α3 454-467 antibodies recognised specifically and in a dose-dependent manner an immunoreactive band. This M_r58,000 immunoreactive species and the N -deglycosylated derivatives were both coincident with the respective homologues found in both calf cerebral cortex membranes and purified receptor preparations. This is the first report of the identification of a specific GABA_A receptor subunit expressed in a human cell line, and it therefore provides a convenient model for the study of receptor structure and regulation.     

Desensitization of CholecystokininB Receptors in GH3 Cells

Abstract: Desensitization of the cholecystokinin (CCK) octapeptide (CCK-8)-induced rise in intracellular free calcium concentration ([Ca²⁺],) was characterized in GH₃ cells, a pituitary tumor cell line, which are known to possess CCK_B receptor subtype. The CCK-8-induced [Ca²⁺], transient was reduced following the initial application of CCK-8. A similar desensitization of the CCK-8-induced response was observed following the first application of thyrotropin-releasing hormone (TRH). By contrast, the TRH- induced response was not desensitized by the preceding application of CCK-8. Desensitization of the CCK-8-induced [Ca²⁺], transient was associated with diminished inositol 1,4,5-trisphosphate formation. The recovery of desensitization of the CCK-8-induced response was delayed by a phosphoserine/phosphothreonine phosphatase inhibitor, calyculin A (100 n M ). The responsiveness to CCK-8 was also reduced by phorbol 12, 13-dibutyrate (PDBu), and this effect of PDBu was completely abolished by preincubation with staurosporine. Staurosporine significantly attenuated the desensitization caused by preincubation with CCK-8, but this effect was too small to attribute the desensitization to the protein kinase C transduction pathway alone. It is likely that desensitization of CCK receptors involves multiple transduction pathways.     

Activation of Phosphodiesterase IV During Desensitization of the A2A Adenosine Receptor-Mediated Cyclic AMP Response in Rat Pheochromocytoma (PC12) Cells

Abstract: Prolonged activation of an A_2A adenosine receptor significantly inhibits the cellular response to subsequent stimulation (A_2A desensitization). We have reported previously that activation of phosphodiesterase (PDE) contributes to A_2A desensitization in PC12 cells. In the present study, we show that a type IV PDE (PDE4)-selective inhibitor (Ro 20-1724) effectively blocks the increase in PDE activity in desensitized cells. Thus, PDE4 appears to be the PDE specifically activated during A_2A desensitization in PC12 cells. Prolonged treatment of PC12 cells with an A_2A-selective agonist (CGS21680) leads to increased PDE4 activity in a dose-dependent manner, which can be blocked by an A_2A-selective antagonist [8-(3-chlorostyryl)caffeine]. Using two PDE4 antibodies, we were able to demonstrate that the levels of two PDE4-immunoreactive bands (72 and 79 kDa) were increased significantly during A_2A desensitization. Prolonged treatment with forskolin to elevate intracellular cyclic AMP contents also resulted in increased PDE4 activity. In addition, activation of PDE4 activity during A_2A desensitization could be blocked by a protein kinase A (PKA)-selective inhibitor (H89) and was not observed in a PKA-deficient PC12 cell line (A123). Taken together, activation of PDE4 via a cyclic AMP/PKA-dependent pathway plays a critical role in dampening the signal of the A_2A receptor.     

Inhibition of Bradykinin-Induced Cytosolic Ca2+ Elevation by Muscarinic Stimulation Without Attenuation of Inositol 1,4,5-Trisphosphate Production in Human Neuroblastoma SK-N-BE(2)C Cells

Abstract: Cross talk between two phospholipase C (PLC)-linked receptor signalings was investigated in SK-N-BE(2)C human neuroblastoma cells. Sequential stimulation with two agonists at 5-min intervals was performed to examine the interaction between muscarinic and bradykinin (BK) receptors. Pretreatment of cells with a maximal effective concentration (5 µ M ) of BK did not affect the subsequent carbachol (CCh)-induced [Ca²⁺]_i rise, but CCh (1 m M ) pretreatment completely abolished the BK-induced [Ca²⁺]_i rise without inhibition of BK-induced inositol 1,4,5-trisphosphate (IP₃) generation. Thapsigargin (1 µ M ) pretreatment abolished the subsequent BK- and CCh-induced [Ca²⁺]_i rise, though it did not affect agonist-induced IP₃ generation. However, the addition of atropine at plateau phases of CCh-induced [Ca²⁺]_i rise and IP₃ production caused a rapid decline to the basal levels and then restored the [Ca²⁺]_i rise by BK. Treatment of cells with both CCh and BK at the same time showed additive effects in IP₃ production. However, the [Ca²⁺]_i rise induced by both agonists in the presence or absence of extracellular Ca²⁺ was the same as the responses triggered by CCh alone. The results suggest that each receptor or receptor-linked PLC activity is not influenced by pretreatment with the other agonist but IP₃-sensitive Ca²⁺ stores are shared by signal pathways from both receptors.     

The δ-Opioid Receptor in SK-N-BE Human Neuroblastoma Cell Line Undergoes Heterologous Desensitization

Abstract: The human neuroblastoma cell line SK-N-BE expresses δ-opioid receptors negatively coupled to adenylyl cyclase. Prolonged treatment (2 h) of the cells with 100 n M etorphine leads to an almost complete desensitization (8.2 ± 5.9 vs. 45.8 ± 8.7% for the control). Other receptors negatively coupled to adenylyl cyclase, namely, D2-dopaminergic, α₂-adrenergic, and m2/m4-muscarinic, were identified by screening of these cells, and it was shown that prolonged treatment (2 h) with 1 µ M 2-bromo-α-ergocryptine or 1 µ M arterenol resulted in a marked desensitization of D2-dopaminergic and α₂-adrenergic receptors, respectively. Cross-desensitization experiments revealed that pretreatment with etorphine desensitized with the same efficiency the δ-opioid receptor and the D2-dopaminergic receptor, and pretreatment with 2-bromo-α-ergocryptine also desensitized both receptors. In contrast, pretreatment with etorphine desensitized only partly the α₂-adrenergic receptor response, whereas pretreatment with 1 µ M arterenol partly desensitized the δ-opioid receptor response. It is concluded that the δ-opioid receptor-mediated inhibitory response of adenylyl cyclase undergoes heterologous desensitization, and it is suggested that δ-opioid and D2-dopaminergic receptors are coupled to adenylyl cyclase via a G_i2 protein, whereas α₂-adrenergic receptor could be coupled to the enzyme via two G proteins, G_i2 and another member of the G_i/G_o family.     

CCK receptor phosphorylation exposes regulatory domains affecting phosphorylation and receptor trafficking

Agonist-stimulatedphosphorylation of guanine nucleotide-binding protein (Gprotein)-coupled receptors has been recognized as an importantmechanism for desensitization by interfering with coupling of theactivated receptor with its G protein. We recently described a mutantof the CCK receptor that modified two of five key sites ofphosphorylation (S260,264A) and eliminated agonist-stimulated receptorphosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant hadimpaired rapid desensitization but was ultimately able to bedesensitized by normal receptor internalization. Here we demonstratethat this mutant receptor is also defective in resensitization, withabnormal recycling to the cell surface. To explore this, anotherreceptor mutant was prepared, replacing the same serines withaspartates to mimic the charge of serine-phosphate (S260,264D). Thismutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. Itwas internalized like wild-type receptors and was resensitized andtrafficked normally. This provides evidence for an additional importantfunction for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor toexpose other potential sites of phosphorylation and to expose domainsinvolved in the targeting and trafficking of endosomes. Thehierarchical phosphorylation of these sites may play a key role inreceptor regulation.

     

Effect of Heat Shock on Intracellular Calcium Mobilization in Neuroblastoma × Glioma Hybrid Cells

Abstract: The effect of heat shock on agonist-stimulated intracellular Ca²⁺ mobilization and the expression of heat shock protein 72 (hsp72) in neuroblastoma × glioma hybrid cells (NG 108–15 cells) were examined. Hsp72 was expressed at 6 h after heat shock (42.5°C, 2 h), reached a maximum at 12 h, and decreased thereafter. Bradykinin-induced [Ca²⁺], rise was attenuated to 28% of control by heat shock at 2 h after heat shock, and reversion to the control level was seen 12 h later. When the cells were treated with quercetin or antisense oligodeoxyribonucleotide against hsp72 cDNA, the synthesis of hsp72 was not induced by heat shock, whereas bradykinin-induced [Ca²⁺]_i rise was abolished and the [Ca²⁺]_i rise was not restored. Recovery from this stressed condition was evident when cells were stimulated by the Ca²⁺-ATPase inhibitor thapsigargin, even in the presence of either quercetin or antisense oligodeoxyribonucleotide. Inositol 1,4,5-trisphosphate (IP₃) production was not altered by heat shock at 12 h after heat shock, whereas IP₃ receptor binding activity was reduced to 45.3%. In the presence of quercetin or antisense oligodeoxyribonucleotide, IP₃ receptor binding activity decreased and reached 27.2% of the control 12 h after heat shock. Our working thesis is that heat shock transiently suppresses the IPs-mediated intracellular Ca²⁺ signal transduction system and that hsp72 is involved in the recovery of bradykinin-induced [Ca²⁺]_i rise.     

Specific Receptor-Mediated Inhibition of Cyclic AMP Synthesis by Dopamine in a Neuroblastoma × Brain Hybrid Cell Line NCB-20

Abstract: Dopamine and dopamine receptor agonists were found to inhibit adenylate cyclase activity dose-de-β ndently in a neuroblastoma × Chinese hamster brain explant hybrid cell line NCB-20. Apomorphine (with an IC₅₀ value of 10 n M ) was the most effective inhibitor, followed by 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydro-naphthaline (ADTN), dopamine, and N -dipropyldopa-mine. The inhibition was potently reversed by sulpiride, butaclamol, and flupenthixol in a stereospecific manner, but was unaffected by yohimbine, except at high concentrations. Clonidine also inhibited adenylate cyclase activity in these cells and this was reversed by the α₂-adrenoreceptor antagonist yohimbine, but not by sulpiride. [ d -Ala², d -Leu⁵]Enkephalin inhibited adenylate cyclase activity in NCB-20 cells at nanomolar concentrations; this was reversed by naloxone. All three inhibitory neurotransmitters were able to reverse the stimulation of cyclic AMP synthesis by serotonin or prostaglandin E₁The dopamine receptor that modulates cyclic AMP synthesis in NCB-20 cells is pharmacologically quite distinct from a high-affinity spiperone binding site identified in these cells, but shows the pharmacologic specificity of the D₂ receptor previously described in mammalian brain.     

Involvement of Both Inhibitory and Stimulatory Guanine Nucleotide Binding Proteins in the Expression of Chronic Opiate Regulation of Adenylate Cyclase Activity in NG108-15 Cells

Abstract: Chronic etorphine treatment of neuroblastoma × glioma NG108-15 cells results in both an increase in adenylate cyclase activity (upon addition of the opiate antagonist naloxone) as well as an homologous desensitization of the opiate receptor. The continued ability of opiate agonists to regulate adenylate cyclase activity following opiate receptor desensitization can be understood by proposing that the catalytic subunit of adenylate cyclase in NG108-15 cells is under tonic regulation by both guanine nucleotide regulatory (N_i) and stimulatory (N_s) components. Inactivation of N_i by pertussis toxin (PT) treatment resulted in elevated adenylate cyclase activities comparable to those observed in control cells following chronic opiate treatment. This increased enzymatic activity could not be further induced by PT treatment of cells exposed to opiate previously. In addition, procedures that prevented receptor-mediated activation of N_s, i.e., treatment with NaF or desensitization of the stimulatory receptors (prostaglandin E₁, adenosine) eliminated the increase in adenylate cyclase activity induced by naloxone following chronic opiate exposure. Hence, the increase in enzymatic activity observed following chronic opiate treatment may be due to a loss in tonic inhibitory regulation of adenylate cyclase mediated through N_i resulting in the unimpeded expression of N_s activity. This tonic inhibition of adenylate cyclase activity is one of the multiple mechanisms by which N_i regulates adenylate cyclase in this cell line.     

Glycogen Synthase Kinase-3 modulates α1A-adrenergic receptor action and regulation

The possibility that glycogen synthase kinase 3 (GSK3) could modulate α_1A-adrenergic receptor (α_1A-AR) function and regulation was tested employing LNCaP and HEK293 cells transfected to express the enhanced green fluorescent protein-tagged human α_1A-AR. Receptor phosphorylation and internalization, intracellular free calcium, α_1A-AR-GSK3 colocalization, and coimmunoprecipitation were studied. The effects of the pharmacological GSK3 inhibitor, SB-216763, and the coexpression of a dominant-negative mutant of this kinase, as well as the signaling, desensitization, and internalization of receptors with S229, S258, S352, and S381 substitutions for alanine or aspartate, were also determined. SB-216763 inhibited agonist- and phorbol myristate acetate (PMA)-mediated α_1A-AR phosphorylation, reduced oxymetazoline-induced desensitization, and magnified that induced by PMA. Agonists and PMA increased receptor-GSK3 colocalization and coimmunoprecipitation. Expression of a dominant-negative GSK3 mutant reduced agonist- but not PMA-induced receptor internalization. α_1A-AR with the GSK3 putative target sites mutated to alanine exhibited reduced phosphorylation and internalization in response to agonists and increased PMA-induced desensitization. Agonist-induced, but not PMA-induced, receptor-β arrestin intracellular colocalization was diminished in cells expressing the GSK3 putative target sites mutated to alanine. Our data indicated that GSK3 exerts a dual action on α_1A-AR participating in agonist-mediated desensitization and internalization and avoiding PMA-induced desensitization.     

Regulation of Corticotropin-Releasing Factor Receptor Function in Human Y-79 Retinoblastoma Cells: Rapid and Reversible Homologous Desensitization but Prolonged Recovery

Abstract: Homologous receptor desensitization is an important regulatory response to continuous activation by agonist that involves the uncoupling of a receptor from its G protein. When human retinoblastoma Y-79 cells expressing corticotropin-releasing factor (CRF) receptors were preincubated with CRF for 10 min-4 h, a time-dependent reduction in both the peak and sensitivity of CRF-stimulated intracellular cyclic AMP (cAMP) accumulation developed with a t _1/2 of 38 min and an EC₅₀ of 6–7 n M CRF. CRF receptor desensitization was slowly reversible after a 4-h CRF preincubation with a t _1/2 of 13 h and a full restoration of cAMP responsiveness to CRF at 24 h following the removal of 10 n M CRF. Because the ability of vasoactive intestinal peptide, forskolin, or (−)-isoproterenol to stimulate cAMP accumulation was not diminished in Y-79 cells desensitized with 10 n M CRF, the observed desensitization was considered to be a specific homologous action of CRF. CRF receptor desensitization was markedly attenuated by CRF receptor antagonists, which alone did not produce any appreciable reduction in CRF-stimulated cAMP accumulation. Although recent reports have demonstrated a rapid decline in steady-state levels of CRF receptor type 1 (CRF-R1) mRNA in anterior pituitary cells during several hours of exposure to CRF, there was no observed reduction in CRF-R1 mRNA levels when Y-79 cells were preincubated with 10 n M CRF for 10 min-24 h despite a rapid time- and concentration-dependent loss of CRF receptors from the retinoblastoma cell surface.     

Regulation of membrane cholecystokinin-2 receptor by agonists enables classification of partial agonists as biased agonists

Given the importance of G-protein-coupled receptors as pharmacological targets in medicine, efforts directed at understanding the molecular mechanism by which pharmacological compounds regulate their presence at the cell surface is of paramount importance. In this context, using confocal microscopy and bioluminescence resonance energy transfer, we have investigated internalization and intracellular trafficking of the cholecystokinin-2 receptor (CCK2R) in response to both natural and synthetic ligands with different pharmacological features. We found that CCK and gastrin, which are full agonists on CCK2R-induced inositol phosphate production, rapidly and abundantly stimulate internalization. Internalized CCK2R did not rapidly recycle to plasma membrane but instead was directed to late endosomes/lysosomes. CCK2R endocytosis involves clathrin-coated pits and dynamin and high affinity and prolonged binding of β-arrestin1 or -2. Partial agonists and antagonists on CCK2R-induced inositol phosphate formation and ERK1/2 phosphorylation did not stimulate CCK2R internalization or β-arrestin recruitment to the CCK2R but blocked full agonist-induced internalization and β-arrestin recruitment. The extreme C-terminal region of the CCK2R (and more precisely phosphorylatable residues Ser(437)-Xaa(438)-Thr(439)-Thr(440)-Xaa(441)-Ser(442)-Thr(443)) were critical for β-arrestin recruitment. However, this region and β-arrestins were dispensable for CCK2R internalization. In conclusion, this study allowed us to classify the human CCK2R as a member of class B G-protein-coupled receptors with regard to its endocytosis features and identified biased agonists of the CCK2R. These new important insights will allow us to investigate the role of internalized CCK2R·β-arrestin complexes in cancers expressing this receptor and to develop new diagnosis and therapeutic strategies targeting this receptor.     

Regulation of CB1 cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism

Agonists stimulate cannabinoid 1 receptor (CB₁R) internalization. Previous work suggests that the extreme carboxy-terminus of the receptor regulates this internalization – likely through the phosphorylation of serines and threonines clustered within this region. While truncation of the carboxy-terminus (V460Z CB₁) and consequent removal of these putative phosphorylation sites prevents endocytosis in AtT20 cells, the residues necessary for CB₁R internalization remain elusive. To determine the structural requirements for internalization, we evaluated endocytosis of carboxy-terminal mutant CB₁Rs stably expressed in HEK293 cells. In contrast to AtT20 cells, V460Z CB₁R expressed in HEK293 cells internalized to the same extent and with similar kinetics as the wild-type receptor. However, mutation of serine and/or threonine residues within the extreme carboxy-terminal attenuated internalization when these receptors were expressed in HEK293 cells. These results establish that the extreme carboxy-terminal phosphorylation sites are not required for internalization of truncated receptors, but are required for internalization of full-length receptors in HEK293 cells. Analysis of β-arrestin-2 recruitment to mutant CB₁R suggests that putative carboxy-terminal phosphorylation sites mediate β-arrestin-2 translocation. This study indicates that the local cellular environment affects the structural determinants of CB₁R internalization. Additionally, phosphorylation likely regulates the internalization of (full-length) CB₁Rs.     

Inositol 1,4,5-trisphosphate formation in leaves of winter oilseed rape plants in response to freezing, tissue water potential aed abscisic acid

Time courses of formation of inositol 1,4,5-trisphosphate (IP₃) were followed in the leaves of non-acclimated and cold (2°C)-acclimated winter oilseed rape ( Brassica napus L. var. oleifera ) plants, subjected to different freezing temperatures or to polyethylene glycol 8000 (PEG) and abscisic acid (ABA) treatments. Changes in water potential (Ψ_w) and in ABA level in the frost- and PEG-treated tissues were also determined. Results obtained indicate that temperatures sligthly higher than LT₅₀ induced a transient and substantial increase in IP₃ level, both in non-acclimated and cold-acclimated tissues. At comparable freezing temperature (–5°C) the response of cold-acclimated leaves was lower than that of non-acclimated ones. The PEG-depedent decrease in Ψ_w to –0.9 MPa or ABA (0.1 m M ) treatment gave rise to a transient increase in IP₃ content in non-acclimated tissues only. Collectively, the data indicate that cold acclimation of plants may lead to lower cell responsiveness to the factors studied in terms of induction of IP₃ formation. Changes in the IP₃ content, observed in the present experiments, support our previous suggestion that non-killing freezing temperatures may induce the phosphoinositide pathway, both in non-acclimated and cold-acclimated tissues. Lowering of tissue water potential to some threshold value or a high exogenous ABA supply may mimic the freezing-dependent reaction in the non-acclimated leaves.