Cerebroventricular administration of somatostatin (SRIF): effect on central levels of cyclic AMP

The changes in cAMP levels in the hippocampus, cerebral cortex and the neostriatum were investigated at different times after the cerebroventricular administration of SRIF. An early significant increase in cAMP levels (at 5 minutes) in the hippocampus induced by SRIF was eliminated by pretreatment with sotalol. However, the overall behavioral response to SRIF was not affected by sotalol. Sotalol itself significantly reduced cAMP levels in control experiments. In cerebral cortex, an SRIF-induced increase in cAMP levels was significantly lowered by sotalol pretreatment at both 5 and 15 minutes post-SRIF. In neostriatum, a sustained elevation in cAMP levels was observed at 5 and 15 minutes after the intraventricular infusion of SRIF. Sotalol pretreatment failed to reduce the cAMP levels although it lowered its increase at 15 min. post-SRIF. The results appear to show a beta adrenergic involvement in the cAMP response to SRIF and an apparent independence of the behavioral response from cAMP changes.     

Structural analysis and functional role of the carbohydrate component of somatostatin receptors.

SRIF receptors are membrane-bound glycoproteins. To structurally identify the carbohydrate components of SRIF receptors, solubilized rat brain SRIF receptors were subjected to lectin affinity chromatography. Solubilized SRIF receptors specifically bound to wheat germ agglutinin-lectin affinity columns but not to succinylated wheat germ agglutinin. This finding, as well as the ability of the solubilized receptor to interact with a Sambucus nigra L. lectin affinity column suggested that sialic acid residues are associated with SRIF receptors. The inability of the receptor to bind to concanavalin A, Dolichus biflorus agglutinin, Ulex europeaus I, and Jacalin lectin affinity columns suggests that high mannose, N-acetylgalactosamine, fucose, and O-linked carbohydrates are not associated with receptor. To investigate the functional role of the carbohydrate groups in brain SRIF receptors, specific sugars were selectively cleaved from SRIF receptors and the subsequent effect on the specific high affinity binding of the agonist [125I]MK 678 to SRIF receptors was determined. Treatment of the receptor with endoglycosidase D did not affect the specific binding of [125I] MK 678 to the solubilized SRIF receptors, consistent with the finding from lectin affinity chromatography that high mannose-type carbohydrate structures were not associated with SRIF receptors. Treatment of solubilized SRIF receptors with peptide-N-glycosidase F and endoglycosidases H and F reduced [125I]MK 678 binding to SRIF receptors indicating that either hybrid, or a combination of hybrid and complex N-linked carbohydrate structures, have a role in maintaining the receptor in a high affinity state for agonists. Treatment of solubilized SRIF receptors with neuraminidase from Vibrio cholera abolished high affinity agonist binding to the receptors, whereas treatment of the receptor with neuraminidase from Newcastle disease virus did not affect [125I]MK 678 binding to the receptor. These findings suggest that sialic acid residues in an alpha 2,6-configuration have a role in maintaining the SRIF receptor in a high affinity conformation for agonists. This is further indicated by studies on SRIF receptors in the pituitary tumor cell line, AtT-20. Treatment of AtT-20 cells in culture with neuraminidase (V. cholera) greatly reduces high affinity [125I] MK 678 binding sites, but did not alter the maximal ability of SRIF to inhibit forskolin-stimulated cAMP accumulation in intact AtT-20 cells. This finding suggests that the desialylated SRIF receptor is functionally active and remains coupled to GTP-binding proteins, but exhibits a reduced affinity for agonists. Treatment of AtT-20 cell membranes with neuraminidase from V. cholera was also able to greatly reduce the affinity of SRIF receptors for [125I]MK 678.(ABSTRACT TRUNCATED AT 400 WORDS)     

Receptor-bound somatostatin and epidermal growth factor are processed differently in GH4C1 rat pituitary cells

GH4C1 cells, a clonal strain of rat pituitary tumor cells, have high-affinity, functional receptors for the inhibitory hypothalamic peptide somatostatin (SRIF) and for epidermal growth factor (EGF). In this study we have examined the events that follow the initial binding of SRIF to its specific plasma membrane receptors in GH4C1 cells and have compared the processing of receptor-bound SRIF with that of EGF. When cells were incubated with [125I-Tyr1]SRIF at temperatures ranging from 4 to 37 degrees C, greater than 80% of the specifically bound peptide was removed by extraction with 0.2 M acetic acid, 0.5 M NaCl, pH 2.5. In contrast, the subcellular distribution of receptor-bound 125I-EGF was temperature dependent. Whereas greater than 95% of specifically bound 125I-EGF was removed by acid treatment after a 4 degrees C binding incubation, less than 10% was removed when the binding reaction was performed at 22 or 37 degrees C. In pulse-chase experiments, receptor-bound 125I-EGF was transferred from an acid-sensitive to an acid-resistant compartment with a half-time of 2 min at 37 degrees C. In contrast, the small amount of [125I-Tyr1]SRIF that was resistant to acid treatment did not increase during a 2-h chase incubation at 37 degrees C. Chromatographic analysis of the radioactivity released from cells during dissociation incubations at 37 degrees C showed that greater than 90% of prebound 125I-EGF was released as 125I-tyrosine, whereas prebound [125I-Tyr1]SRIF was released as a mixture of intact peptide (55%) and 125I-tyrosine (45%). Neither chloroquine (0.1 mM), ammonium chloride (20 mM), nor leupeptin (0.1 mg/ml) increased the amount of [125I-Tyr1]SRIF bound to cells at 37 degrees C. Furthermore, chloroquine and leupeptin did not alter the rate of dissociation or degradation of prebound [125I-Tyr1]SRIF. In contrast, these inhibitors increased the amount of cell-associated 125I-EGF during 37 degrees C binding incubations and decreased the subsequent rate of release of 125I-tyrosine. The results presented indicate that, as in other cell types, EGF underwent rapid receptor-mediated endocytosis in GH4C1 cells and was subsequently degraded in lysosomes. In contrast, SRIF remained at the cell surface for several hours although it elicits its biological effects within minutes. Furthermore, a constant fraction of the receptor-bound [125I-Tyr1]SRIF was degraded at the cell surface before dissociation. Therefore, after initial binding of [125I-Tyr1]SRIF and 125I-EGF to their specific membrane receptors, these peptides are processed very differently in GH4C1 cells.     

Somatostatin pretreatment facilitates GRF-induced GH release and increase in free calcium in pituitary cells

Somatostatin pretreatment sensitizes rat anterior pituitary to hGRF stimulation in vitro. The pretreatment (1 nM for 10 min) facilitated GH release response of dispersed rat anterior pituitary cells to hGRF (1 nM for 3 min) 2.04-fold in a perifusion system. The effect lasted even 20 min after the pretreatment. SRIF pretreatment decreased cAMP content in the cells after hGRF stimulation to 61% of the control value. When hGRF was replaced by 1 mM DBcAMP and 15 mM KCl, the pretreatment increased GH secretion 1.69- and 1.67-fold respectively. SRIF pretreatment (1 nM for 10 min) caused a larger increase in (Ca2+)i by hGRF than that of control. The effect of SRIF pretreatment facilitates GRF-induced increase in GH secretion probably through the stimulation of increase in (Ca2+)i.     

Role of amphiphysin II in somatostatin receptor trafficking in neuroendocrine cells

Amphiphysins are SH3 domain-containing proteins thought to function in clathrin-mediated endocytosis. To investigate the potential role of amphiphysin II in cellular trafficking of G protein-coupled somatostatin (SRIF) receptors, we generated an AtT-20 cell line stably overexpressing amphiphysin IIb, a splice variant that does not bind clathrin. Endocytosis of (125)I-[d-Trp(8)]SRIF was not affected by amphiphysin IIb overexpression. However, the maximal binding capacity (B(max)) of the ligand on intact cells was significantly lower in amphiphysin IIb overexpressing than in non-transfected cells. This difference was no longer apparent when the experiments were performed on crude cell homogenates, suggesting that amphiphysin IIb overexpression interferes with SRIF receptor targeting to the cell surface and not with receptor synthesis. Accordingly, immunofluorescence experiments demonstrated that, in amphiphysin overexpressing cells, sst(2A) and sst(5) receptors were segregated in a juxtanuclear compartment identified as the trans-Golgi network. Amphiphysin IIb overexpression had no effect on corticotrophin-releasing factor 41-stimulated adrenocorticotropic hormone secretion, suggesting that it is not involved in the regulated secretory pathway. Taken together, these results suggest that amphiphysin II is not necessary for SRIF receptor endocytosis but is critical for its constitutive targeting to the plasma membrane. Therefore, amphiphysin IIb may be an important component of the constitutive secretory pathway.     

Insulin-like growth factor-I is internalized after binding to the type I insulin-like growth factor receptor

Receptor-mediated endocytosis may represent an important mechanism whereby peptide hormones exert their biological effects. The ability of recombinant insulin-like growth factor (IGF)-I to be internalized by cultured cells was evaluated in BRL-3A2 cells, a rat liver-derived cell line which lacks insulin receptors. Since recombinant IGF-I does not bind to the Type II IGF receptor, all specific binding of 125I-IGF-I in BRL-3A2 cells represents binding to the Type I receptor. Exposure of BRL-3A2 cells to IGF-I resulted in a rapid 50% downregulation of Type I IGF receptors. Only one-half of these binding sites were sensitive to treatment with trypsin, a phenomenon which indicates that the peptide and its receptor were internalized after the cells were exposed to IGF-I. In conclusion, these experiments demonstrate that IGF-I can be internalized by cultured cells via the Type I IGF receptor, and suggest that IGF hormone action may be exerted by receptor-mediated endocytosis.     

Somatostatin decreases voltage-gated Ca2+ currents in GH3 cells through activation of somatostatin receptor 2

The secretion of growth hormone (GH) is inhibited by hypothalamic somatostatin (SRIF) in somatotropes through five subtypes of the somatostatin receptor (SSTR1-SSTR5). We aimed to characterize the subtype(s) of SSTRs involved in the Ca2+ current reduction in GH3 somatotrope cells using specific SSTR subtype agonists. We used nystatin-perforated patch clamp to record voltage-gated Ca2+ currents, using a holding potential of -80 mV in the presence of K+ and Na+ channel blockers. We first established the presence of T-, L-, N-, and P/Q-type Ca2+ currents in GH3 cells using a variety of channel blockers (Ni+, nifedipine, omega-conotoxin GVIA, and omega-agatoxin IVA). SRIF (200 nM) reduced L- and N-type but not T- or P/Q-type currents in GH3 cells. A range of concentrations of each specific SSTR agonist was tested on Ca2+ currents to find the maximal effective concentration. Activation of SSTR2 with 10(-7) and 10(-8) M L-797,976 decreased the voltage-gated Ca2+ current and abolished any further decrease by SRIF. SSTR1, SSTR3, SSTR4, and SSTR5 agonists at 10(-7) M did not modify the voltage-gated Ca2+ current and did not affect the Ca2+ current response to SRIF. These results indicate that SSTR2 is involved mainly in regulating voltage-gated Ca2+ currents by SRIF, which contributes to the decrease in intracellular Ca2+ concentration and GH secretion by SRIF.     

Purification of a pituitary receptor for somatostatin. The utility of biotinylated somatostatin analogs.

A somatostatin (SRIF) receptor and its associated Gi regulatory proteins was purified from GH4C1 rat pituitary cells by: 1) saturation of the membrane-bound receptor with biotinyl-NH-[Leu8,D-Trp22,Tyr25] SRIF28 (bio-S28); 2) solubilization of receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine (D.L); 3) adsorption of solubilized receptor-ligand complex to immobilized streptavidin; and 4) elution of receptor and G-protein by GTP. The receptor, a glycoprotein with an average M(r) of 85,000, was then purified to substantial homogeneity on immobilized wheat germ agglutinin. The 85-kDa glycoprotein was identified as a SRIF receptor by several criteria. (a) It had the same size as the chemically cross-linked R.[125I]L complex. (b) Yield of the purified protein increased and plateaued in the same range of bio-S28 concentrations where specific high affinity binding reached saturation. (c) It was copurified with appropriate G-protein subunits. The 85-kDa receptor and two other proteins with M(r) values of 35,000 and 40,000, the sizes of G beta and G alpha, did not appear in eluates from control streptavidin columns done with SRIF receptors loaded with nonbiotinylated S14. The 40-kDa protein was identified as a Gi alpha by ADP-ribosylation from [32P]NAD catalyzed by pertussis toxin. (d) Both the chemically cross-linked R.[125I]L complex and SRIF receptor purified from [35S]methionine-labeled GH4C1 cells were reduced in size to about 38 kDa by endoglycosidase F. (e) Amino acid sequence from the purified receptor was nearly identical with that of a recently cloned SRIF receptor subtype.     

Identification of the subunits of GTP-binding proteins coupled to somatostatin receptors.

Somatostatin (SRIF) induces its biological effects by interacting with membrane-bound receptors that are linked to cellular effector systems via G proteins. We have studied SRIF receptor-G protein associations by solubilizing the SRIF receptor from rat brain and AtT-20 cells and immunoprecipitating the receptor-G protein complex with peptide-directed antisera against the different subunits of the G protein heterotrimer. Antiserum 8730, which selectively interacts with all Gi alpha subtypes, maximally and specifically immunoprecipitated SRIF receptor-Gi alpha complexes. To identify the subtypes of Gi alpha that are coupled to SRIF receptors, the subtype-selective antisera 3646, 1521, and 1518, which specifically interact with Gi alpha 1, Gi alpha 2, and Gi alpha 3, respectively, were used to immunoprecipitate SRIF receptor-Gi alpha complexes. Antiserum 3646 immunoprecipitated SRIF receptor-Gi alpha 1 complexes from both brain and AtT-20 cells. Antiserum 1521 immunoprecipitated Gi alpha 2 from both brain and AtT-20 cells but did not immunoprecipitate SRIF receptors from these tissues. Antiserum 1518 immunoprecipitated AtT-20 cell SRIF receptors but uncoupled brain SRIF receptor-G protein complexes. This result was confirmed with another peptide-selective antiserum, SQ, directed against Gi alpha 3. The findings from these studies indicate that Gi alpha 1 and Gi alpha 3 are coupled to SRIF receptors, whereas Gi alpha 2 is not. Even though brain and AtT-20 cell SRIF receptors were both coupled to Gi alpha, the receptors from these tissues differed in their coupling to Go alpha. Antiserum 2353, which is directed against Go alpha, immunoprecipitated SRIF receptors from AtT-20 cells, but did not immunoprecipitate or uncouple SRIF receptor-G protein complexes from rat brain. To determine the beta subunits associated with the SRIF receptor, antisera directed against G beta 36 and G beta 35 were used to immunoprecipitate SRIF receptor-G protein complexes from brain. Peptide-directed antiserum against G beta 36 selectively immunoprecipitated solubilized brain SRIF receptors. However, antiserum directed against the G beta 35 subunit did not immunoprecipitate brain SRIF receptors, suggesting that brain SRIF receptors may preferentially associate with G beta 36. In addition to coimmunoprecipitating with Gi alpha and G beta, brain SRIF receptors coimmunoprecipitated the G protein gamma subunits, G gamma 2 and G gamma 3. These results provide the first evidence that SRIF receptors are coupled to different subunits of G proteins and suggest that selectivity exists in the association of different G protein subunits with the SRIF receptor.     

Both somatostatin and the caudal neuropeptide, urotensin II, stimulate lipid mobilization from coho salmon liver incubated in vitro

The direct effects of somatostatin-14 (SRIF; synthetic ovine) and the fish caudal neuropeptide, urotensin II (UII; synthetic Gillichthys), on fatty acid (FA) release and on lipolytic enzyme (triacylglycerol lipase) activity were determined on coho salmon liver slices incubated in vitro. FA release was continuously measured by pH-stat titration. Additionally, gas chromatographic analysis of the incubation medium was performed to determine the type and relative composition of medium fatty acid constituents. SRIF and UII both stimulated FA release in a dose-dependent manner; the two peptides appeared to stimulate FA release in an equimolar manner. Maximal response was obtained at 1 X 10(-5) M; ED50 was approximately 2 X 10(-7) M. SRIF-stimulated FA release did not result in differential secretion of any particular FA type. Tissue triacylglycerol lipase activity was significantly enhanced by addition of UII or SRIF (2 X 10(-6) M). Dibutyryl cAMP and IBMX both stimulated FA release and lipase activity; dbcAMP stimulated FA release in dose-dependent manner. These results indicate that SRIF and UII directly enhance lipid mobilization from salmon liver slices and suggest that SRIF- and UII-stimulated lipid mobilization from salmon liver slices is mediated through cAMP.     

Expression of haptoglobin receptors in human hepatoma cells.

The uptake of radio-labeled hemoglobin-haptoglobin complex (Hb-Hp) by human hepatoma PLC/PRF/5 and HepG2 cells was investigated in an attempt to characterize the uptake process and intracellular transport. Human hepatoma cells took up Hb-Hp in a receptor-mediated manner. Scatchard analysis of binding revealed that PLC/PRF/5 and HepG2 cells exhibited about 21,000 and 63,000 haptoglobin receptors/cell, with a dissociation constant (Kd) of 8.0 and 17 nM, respectively. Human hepatocytes in primary culture also expressed about 84,000 receptors/cells, with a Kd of 7.4 nM. The hemoglobin-haptoglobin complex was internalized and subsequently the internalized Hb-Hp was slowly degraded in the cells. Preincubation of the cells with Hb-Hp resulted in a decrease in binding of the radioactive Hb-Hp to the cell surface, and was accompanied with an accumulation of intracellular receptors. The uptake of Hb-Hp by the cells was not inhibited by 100 microM chloroquine or by 10 mM methylamine, but was inhibited by 50 microM monodansylcadaverine. Hemoglobin-heme taken up by the cells induced microsomal heme oxygenase. Thus, human hepatoma PLC/PRF/5 and HepG2 cells can take up Hb-Hp by haptoglobin receptor-mediated endocytosis and Hb-Hp probably causes translocation of the haptoglobin receptors from the cell surface to the cell interior where they can be degraded. The internalized heme-moiety of hemoglobin can regulate the expression of heme oxygenase.     

Acute upregulation of interleukin-1 receptor by ligand.

In this study we have investigated the effect that interleukin 1 (IL-1) has on cell surface IL-1 receptor expression in the murine thymoma cell line, EL4 6.1. These cells express IL-1 receptors with both high affinity (Kd = 65 pM, 986 receptors/cell) and low affinity (Kd = 14.5 nM, 10,417 receptors/cell). The high- and low-affinity receptors are indistinguishable by crosslinking studies performed at both high and low ligand concentrations. However, the two affinity states could be functionally distinguished on the basis of their internalization of ligand. Receptor-mediated endocytosis was dependent upon the concentration of ligand bound to the cells. In the presence of low IL-1 concentrations receptor-mediated endocytosis was slow, whereas at high IL-1 concentrations, endocytosis was more rapid. Furthermore, receptor-mediated endocytosis of IL-1 did not result in downregulation of surface IL-1 receptors. Indeed, both kinetic and equilibrium binding studies revealed that pre-incubation of cells with IL-1 alpha resulted in an acute upregulation of 125IL-1 alpha binding to high affinity surface receptors in a time and energy dependent manner. Examination of the association kinetics suggested that increased binding was not attributable to positive co-operativity of the high affinity IL-1 receptor, but was due to increasing IL-1 receptor number. This observation was confirmed by equilibrium binding studies. Moreover, receptor numbers were not enhanced by de novo synthesis, nor release of receptors from an intracellular pool. The observed increases in surface ligand binding were most probably due to conversion of the surface pool of low affinity receptors into high affinity receptors.     

Stimulation of receptor-mediated low density lipoprotein endocytosis in neuraminidase-treated cultured bovine aortic endothelial cells

Sialic acids, occupying a terminal position in cell surface glycoconjugates, are major contributors to the net negative charge of the vascular endothelial cell surface. As integral membrane glycoproteins, LDL receptors also bear terminal sialic acid residues. Pretreatment of near-confluent, cultured bovine aortic endothelial cells (BAEC) with neuraminidase (50 mU/ml, 30 min, 37 degrees C) stimulated a significant increase in receptor-mediated 125I-LDL internalization and degradation relative to PBS-treated control cells. Binding studies at 4 degrees C revealed an increased affinity of LDL receptor sites on neuraminidase-treated cells compared to control BAEC (6.9 vs. 16.2 nM/10(6) BAEC) without a change in receptor site number. This enhanced LDL endocytosis in neuraminidase-treated cells was dependent upon the enzymatic activity of the neuraminidase and the removal of sialic acid from the cell surface. Furthermore, enhanced endocytosis due to enzymatic alteration of the 125I-LDL molecules was excluded. In contrast to BAEC, neuraminidase pretreatment of LDL receptor-upregulated cultured normal human fibroblasts resulted in an inhibition of 125I-LDL binding, internalization, and degradation. Specifically, a significant inhibition in 125I-LDL internalization was observed at 1 hr after neuraminidase treatment, which was associated with a decrease in the number of cell surface LDL receptor sites. Like BAEC, neuraminidase pretreatment of human umbilical vein endothelial cells resulted in enhanced receptor-mediated 125I-LDL endocytosis. These results indicate that sialic acid associated with either adjacent endothelial cell surface molecules or the endothelial LDL receptor itself may modulate LDL receptor-mediated endocytosis and suggest that this regulatory mechanism may be of particular importance to endothelial cells.     

Muscarinic receptor activity change after prolonged treatment with growth hormone and somatostatin

1. The effect of 10-day treatment with growth hormone (GH) (1 mg/kg body weight day) and somatostatin (SRIF) (0.25 mg/kg body weight day) subcutaneously on the activity of muscarinic (M) receptors in rat hypothalamic, pituitary and cerebral cortical membrane fractions was studied using (3H)quinuclidinyl benzylate [(3H)QNB] as radioligand. 2. The administration of GH and SRIF significantly decreased the M-receptor binding affinity in the hypothalamus. 3. In the pituitary the M-receptor affinity was increased after both GH and SRIF treatment. 4. In the hypothalamus and the pituitary the binding capacity of muscarinic receptors was unchanged. 5. In the cerebral cortex the chronical GH injection induced an increase in the number of antagonist binding sites and a decrease of their affinity, while the similar SRIF treatment led to an increase of the binding affinity without any change of M-receptor capacity. 6. These results indicate that GH and SRIF selectively and region-specifically modulate muscarinic receptor binding affinity and capacity and provide new insight into the feedback regulatory mechanisms of GH secretion.     

The influence of mammalian and teleost somatostatins on the secretion of growth hormone from goldfish (Carassius auratus L.) pituitary fragments in vitro

The effect of various vertebrate somatostatins (SRIF) on basal growth hormone (GH) secretion from goldfish pituitary fragments was studied using an in vitro perifusion system. SRIF-14 caused a rapid and dose-dependent decrease in the rate of GH release from goldfish pituitary fragments. The half-maximal effective dose (ED50) of SRIF-14 was calculated as 1.3 nM following exposure to two minute pulses of increasing concentrations of SRIF-14, whereas the ED50 of SRIF-14 calculated after continuous exposure to sequentially increasing doses of SRIF-14 was 65 nM. This difference suggests that the pituitary fragments were less responsive to SRIF-14 in the latter experiment, possibly as a result of previous exposure to SRIF-14. SRIF-28 was found to be equipotent with SRIF-14 in decreasing basal GH secretion from the goldfish pituitary. In contrast, catfish SRIF-22, a uniquely teleost SRIF isolated from catfish pancreatic islets, did not alter GH secretion. These results provide further support for the hypothesis that SRIF-14 or a very similar molecule functions as a GH release-inhibiting factor in teleosts, indicating that this action of SRIF-14 has been fully conserved throughout vertebrate evolution.     

A phosphorylation-regulated brake mechanism controls the initial endocytosis of opioid receptors but is not required for post-endocytic sorting to lysosomes.

The delta-opioid receptor (DOR) can undergo proteolytic down-regulation by endocytosis of receptors followed by sorting of internalized receptors to lysosomes. Although phosphorylation of the receptor is thought to play an important role in controlling receptor down-regulation, previous studies disagree on whether phosphorylation is actually required for the agonist-induced endocytosis of opioid receptors. Furthermore, no previous studies have determined whether phosphorylation is required for subsequent sorting of internalized receptors to lysosomes. We have addressed these questions by examining the endocytic trafficking of a series of mutant versions of DOR expressed in stably transfected HEK 293 cells. Our results confirm that phosphorylation is not required for agonist-induced endocytosis of truncated mutant receptors that lack the distal carboxyl-terminal cytoplasmic domain containing sites of regulatory phosphorylation. However, phosphorylation is required for endocytosis of full-length receptors. Mutation of all serine/threonine residues located in the distal carboxyl-terminal tail domain of the full-length receptor to alanine creates functional mutant receptors that exhibit no detectable agonist-induced endocytosis. Substitution of these residues with aspartate restores the ability of mutant receptors to undergo agonist-induced endocytosis. Studies using green fluorescent protein-tagged versions of arrestin-3 suggest that the distal tail domain, when not phosphorylated, inhibits receptor-mediated recruitment of beta-arrestins to the plasma membrane. Biochemical and radioligand binding studies indicate that, after endocytosis occurs, phosphorylation-defective mutant receptors traffic to lysosomes with similar kinetics as wild type receptors. We conclude that phosphorylation controls endocytic trafficking of opioid receptors primarily by regulating a "brake" mechanism that prevents endocytosis of full-length receptors in the absence of phosphorylation. After endocytosis occurs, subsequent steps of membrane trafficking mediating sorting and transport to lysosomes do not require receptor phosphorylation.     

Measles virus modulates human T-cell somatostatin receptors and their coupling to adenylyl cyclase.

The possible role of immunomodulatory peptide somatostatin (SRIF) in measles virus (MV)-induced immunopathology was addressed by analysis of SRIF receptors and their coupling to adenylyl cyclase in mitogen-stimulated Jurkat T cells and human peripheral blood mononuclear cells (PBMC). SRIF-specific receptors were assayed in semipurified membrane preparations by using SRIF14 containing iodinated tyrosine at the first position in the amino acid chain ([125I]Tyr1) as a radioligand. A determination of receptor number by saturation of radioligand binding at equilibrium showed that in Jurkat cells, MV infection led to a dramatic decrease in the total receptor number. The virus-associated disappearance of one (Ki2 = 12 +/- 4 nM [mean +/- standard error of the mean [SEM]]; n = 4) of two somatostatin binding sites identified in control Jurkat cells (Ki1 = 78 +/- 3 pM and Ki2 = 12 +/- 4 nM [mean +/- SEM]; n = 4) was also observed. Almost identical results were obtained for phytohemagglutinin-activated human PBMC. In the absence of MV infection, two somatostatin binding sites were present (Ki1 = 111 +/- 31 pM and Ki2 = 17 +/- 2 nM [mean +/- SEM]; n = 2), whereas in MV-infected cells, only the high-affinity (Ki1 = 48 +/- 15 pM [mean +/- SEM]; n = 2) binding site remained. In addition, MV infection reinforced the inhibitory effects of SRIF on adenylyl cyclase activity, since maximal inhibition at 1 microM peptide was 11% +/- 4% in control cells versus 25% +/- 3% (P < 0.05) in infected Jurkat cells. Moreover, MV infection severely impaired the capacity of adenylyl cyclase to be activated directly (by forskolin) or indirectly (via Gs protein-coupled vasoactive intestinal peptide receptor). An assessment of [methyl-3H]thymidine incorporation showed that SRIF increased proliferative responses to mitogens only in control cells, not in MV-infected cells. Altogether, our data emphasize that MV-associated alteration of SRIF transduction appears to be related to the loss of SRIF-dependent increase of mitogen-induced proliferation.     

Endocytosis promotes rapid dopaminergic signaling

D(1) dopamine receptors are primary mediators of dopaminergic signaling in the CNS. These receptors internalize rapidly following agonist-induced activation, but the functional significance of this process is unknown. We investigated D(1) receptor endocytosis and signaling in HEK293 cells and cultured striatal neurons using real-time fluorescence imaging and cAMP biosensor technology. Agonist-induced activation of D(1) receptors promoted endocytosis of receptors with a time course overlapping that of acute cAMP accumulation. Inhibiting receptor endocytosis blunted acute D(1) receptor-mediated signaling in both dissociated cells and striatal slice preparations. Although endocytic inhibition markedly attenuated acute cAMP accumulation, inhibiting the subsequent recycling of receptors had no effect. Further, D(1) receptors localized in close proximity to endomembrane-associated trimeric G protein and adenylyl cyclase immediately after endocytosis. Together, these results suggest a previously unanticipated role of endocytosis, and the early endocytic pathway, in supporting rapid dopaminergic neurotransmission.     

Second-messenger regulation of receptor association with clathrin-coated pits: a novel and selective mechanism in the control of CD4 endocytosis.

CD4, a member of the immunoglobulin superfamily, is not only expressed in T4 helper lymphocytes but also in myeloid cells. Receptor-mediated endocytosis plays a crucial role in the regulation of surface expression of adhesion molecules such as CD4. In T lymphocytes p56lck, a CD4-associated tyrosine kinase, prevents CD4 internalization, but in myeloid cells p56lck is not expressed and CD4 is constitutively internalized. In this study, we have investigated the role of cyclic AMP (cAMP) in the regulation of CD4 endocytosis in the myeloid cell line HL-60. Elevations of cellular cAMP were elicited by 1) cholera toxin, 2) pertussis toxin, 3) forskolin and IBMX, 4) NaF, or 5) the physiological receptor agonist prostaglandin E1. All five interventions led to an inhibition of CD4 internalization. Increased cAMP levels did not inhibit endocytosis per se, because internalization of insulin receptors and transferrin receptors and fluid phase endocytosis were either unchanged or slightly enhanced. The mechanism of cAMP inhibition was further analyzed at the ultrastructural level. CD4 internalization, followed either by quantitative electron microscopy autoradiography or by immunogold labeling, showed a rapid and temperature-dependent association of CD4 with clathrin-coated pits in control cells. This association was markedly inhibited in cells with elevated cAMP levels. Thus these findings suggest a second-messenger regulation of CD4 internalization through an inhibition of CD4 association with clathrin-coated pits in p56lck-negative cells.     

Characterization of the cyclic AMP-independent actions of somatostatin in GH cells. I. An increase in potassium conductance is responsible for both the hyperpolarization and the decrease in intracellular free calcium produced by somatostatin

The neuropeptide somatostatin causes membrane hyperpolarization and reduces the intracellular free calcium ion concentration ([Ca2+]i) in GH pituitary cells. In this study, we have used the fluorescent dyes bisoxonol (bis,-(1,3-diethylthiobarbiturate)-trimethineoxonol) and quin2 to elucidate the mechanisms by which these ionic effects are triggered. Addition of 100 nM somatostatin to GH4C1 cells caused a 3.4 mV hyperpolarization and a 26% decrease in [Ca2+]i within 30 s. These effects were not accompanied by changes in intracellular cAMP concentrations and occurred in cells containing either basal or maximally elevated cAMP levels. To determine which of the major permeant ions were involved in these actions of somatostatin, we examined its ability to elicit changes in the membrane potential and the [Ca2+]i when the transmembrane concentration gradients for Na+, Cl-, Ca2+, and K+ were individually altered. Substitution of impermeant organic ions for Na+ or Cl- did not block either the hyperpolarization or the decrease in [Ca2+]i induced by somatostatin. Decreasing extracellular Ca2+ from 1 mM to 250 nM abolished the reduction in [Ca2+]i but did not prevent the hyperpolarization response. These results show that hyperpolarization was not primarily due to changes in the conductances of Na+, Cl-, or Ca2+. Although the somatostatin-induced decrease in [Ca2+]i did require Ca2+ influx, it was independent of changes in Na+ or Cl- conductance. In contrast, elevating the extracellular [K+] from 4.6 to 50 mM completely blocked both the somatostatin-induced hyperpolarization and the reduction in [Ca2+]i. Furthermore, hyperpolarization of the cells with gramicidin mimicked the effect of somatostatin to decrease the [Ca2+]i and prevented any additional effect by the hormone. These results indicate that somatostatin increases a K+ conductance, which hyperpolarizes GH4C1 cells, and thereby secondarily decreases Ca2+ influx. Since the somatostatin-induced decrease in [Ca2+]i is independent of changes in intracellular cAMP levels, it may be responsible for somatostatin inhibition of hormone secretion by its cAMP-independent mechanism.