Lithium inhibits internalization and endosomal processing of both neuropeptide Y (NPY) Y1 and transferrin receptors

Neuroscience Letters 374 (2005) 43–46

Lithium inhibits internalization and endosomal processing of both neuropeptide Y (NPY) Y1 and transferrin receptors Michael S. Parkera , Renu Sahb , Ambikaipakan Balasubramaniamc , Steven L. Parkerd,∗ a

d

Department of Microbiology and Molecular Cell Sciences, University of Memphis, Memphis, TN 38163, USA b Department of Psychiatry, University of Cincinnati, Cincinnati, OH, USA c Department of Surgery, University of Cincinnati, Cincinnati, OH, USA Department of Pharmacology, University of Tennessee Health Science Center, 874 Union Avenue, Memphis, TN 38163, USA Received 18 August 2004; received in revised form 30 September 2004; accepted 8 October 2004

Abstract Low concentrations of Li+ reduce the rate of internalization of neuropeptide Y (NPY) Y1 receptors [M.S. Parker, S.L. Parker, J.K. Kane, Internalization of neuropeptide Y Y1 and Y5 and of pancreatic polypeptide Y4 receptors is inhibited by lithium in preference to sodium and potassium ions, Regul. Pept., 118 (2004) 67–74]. This Li+ -induced decrease in Y1 receptor internalization could be alleviated by Y1 receptor agonists. As shown by fractionation on Percoll gradients, lithium treatment induces a concentration-related decrease of intermediate and higher endosomal densities that contain the internalized Y1 ligand–receptor complex. This indicates an inhibition of endosome processing and maturation. Internalization of human transferrin shows [Li+ ] sensitivity similar to that of the Y1 receptor, and a similar Li+ -induced decrease in endosomal processing. Lithium treatment thus decreases activity of the endosome system shared in the recycling endocytosis of the Y1 and transferrin receptors. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Receptor sequestration; Cationic regulation; Receptor traffic

Attachment of selective agonists to the neuropeptide Y (NPY) Y1 receptor is quite sensitive to monovalent cations [13]. This also predicts monovalent cation sensitivity of the Y1 receptor in signal transduction and cycling. Activity in these receptorlinked processes can be expected especially for Li+ , which can affect both metabolic transducers and effectors, and in particular G-proteins [1,12]. Low concentrations of Li+ were indeed shown recently to reduce the rate of internalization of NPY Y1 and related receptors [16]. The Y1 receptor is mainly internalized by clathrin/dynamin-linked recycling endocytosis [8,14] involving the perinuclear endocytotic compartment [18], similar to endocytosis of the transferrin receptor (see Ref. [17] for a comparison). This points to the possibility of a more general inhibition of endocytosis by Li+ at the level of endosome formation, which involves large GTPases such as dynamins, as well as various small G-proteins, e.g. from Rab ∗

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0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.10.025

[5] and Rho [6] groups. We indeed find that internalization of both the NPY Y1 and the prototypic perinuclear endocytotic compartment-recycling transferrin receptor is inhibited by Li+ at low concentrations, with a reduction in endosomal processing of receptor–agonist complexes. Peptides were supplied by the American Peptide Company (Sunnyvale, CA), and other chemicals by Sigma (St. Louis, MO). The [125 I]-labeled (Leu31 , Pro34 ) human peptide YY (LP-PYY) and porcine peptide YY (pPYY) were obtained from Perkin-Elmer/NEN (Cambridge, MA). Human transferrin was iodinated as described by Karin and Mintz [11]. Human transferrin and (Leu31 , Pro34 ) human peptide YY (LP-PYY) were iodinated by chloramine T method (see Refs. [11,14]). Chinese hamster ovary (CHO) cells stably expressing the cloned guinea pig Y1 receptor [3] were cultured at 250 ␮g/ml geneticin in F12/D-MEM medium (Gibco, Long Island, NY) as described previously [17]. The experimental treatments were done on 48- or 96-well plates, using F12/D-MEM 1:1 medium (Gibco) without antibiotics and fe-

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tal serum, and with added 0.2% protease-free bovine serum albumin. This medium contains 145 mM Na+ and 4.4 mM K+ ions. The labeling with 125 I-tagged peptides was done at 20 pM [125 I]LP-PYY or at 1 nM [125 I]human transferrin, using 1 ␮M non-labeled LP-PYY and 10 ␮M non-labeled human transferrin to define the respective non-specific binding. After incubation and five cold washes, the wells were extracted for 6 min at 0–4 ◦ C with 0.2 M CH3 COOH–0.5 M NaCl (pH 2.6) to dissociate the cell surface-attached agonist peptides (see Refs. [11,17]). The residues were solubilized in 0.1 M NaOH. Radioactivity was measured in a gammascintillation counter. All experiments with cations included a pretreatment in the growth medium for 20–24 h, to allow a full ionic equilibration. At up to 10 mM of any added alkali cation there was no change in cell numbers over 24 h. The pre-equilibration was followed by washing with F12/D-MEM medium containing the same concentration of cations, and incubation with [125 I]-labeled and non-labeled agonists at 37 ◦ C. Percoll gradient centrifugation was done as specified before [17]. Briefly, pro-gradients constituted of 1 ml 60% sucrose, 5 ml 18% and 4 ml 10% Percoll (both in 0.25 M sucrose–0.01 M HEPES (pH 7.4)) were loaded with 1 ml of cell homogenates labeled with iodinated agonists at 1–10 mM of added cations, sedimented for 55 min at 20,000 rpm (58,000 × gmax ) and 5 ◦ C in SW-41Ti rotor (BeckmanSpinco), and divided into 20 fractions before radioactivity counting. Differences between or within treatments were evaluated by Dunnet’s t-tests [23] after a positive analysis of variance. Differences in distribution of tracers among particulate fractions were evaluated in Student’s t-test. Regression parameter estimates were obtained from curve fits with SigmaPlot software (SPSS, Chicago, IL), version 8.0. Control internalization of the Y1-selective agonist [125 I]LP-PYY at 20 pM saturated within 20 min at 37 ◦ C, with a half-period of less than 7 min (Fig. 1A). The ratio of sequestered to surface labeling increased with concentration of non-labeled Y1 agonist to at least 3 nM, and the amount of steady-state internalized tracer increased to about 0.6 nM of total extracellular LP-PYY. The internalization of 1 nM [125 I]human transferrin in CHO half-saturated at about 8 min (Fig. 1B). At 1–10 mM LiCl induced, as expected [14], a concentration-related decrease in internalization of [125 I]LPPYY (Fig. 1A and Fig. 2) of about 45% at 10 mM. Very similar results were obtained with [125 I]porcine peptide YY as the tracer (not shown). There was a decrease in both the amount of the labeled Y1 agonist internalized, and in the ratio of internalized to surface binding, and both changes were apparent at any interval of observation from 2 to 30 min. A similar [Li+ ]-related decrease was found for internalization of [125 I]-labeled human transferrin (Fig. 1B and Fig. 2). Decrease of the steady-state (20–30 min at 37 ◦ C) internalization of [125 I]LP-PYY by Li+ occurred with less than 20% increase in surface binding of the agonist (inset of Fig. 1A).

Fig. 1. Effect of lithium treatment on the internalization of Y1 ligand LPPYY and of transferrin in CHO cells expressing the cloned guinea pig Y1 receptor. The results are averages of three separate measurements, shown with the respective S.E.M. values. All parameters are from non-linear hyperbolic fits. Half-periods of internalization (T/2, min) and maximum binding values (Bmax , fmol ligand internalized per 100,000 cells) are shown ±1 S.E. of the estimates. Insets show the corresponding cell surface binding. (A) Internalization of [125 I]LP-PYY. T/2 and Bmax : no Li+ , 6.4 ± 0.78 and 1.81 ± 0.22; 1 mM Li+ , 12.8 ± 2.4 and 1.6 ± 0.37; 3 mM Li+ , 13.3 ± 2.9 and 1.33 ± 0.25; 10 mM Li+ , 15.9 ± 4 and 1.03 ± 0.39. (B) Internalization of [125 I]human transferrin. T/2 and Bmax : no Li+ , 7.5 ± 0.6 and 1.60 ± 0.05; 1 mM Li+ , 8.6 ± 2.4 and 1.28 ± 0.14; 3 mM Li+ , 10.4 ± 2.2 and 1.23 ± 0.11; 10 mM Li+ , 12.1 ± 2.4 and 1.18 ± 0.10.

No significant internalization change was noted at 10 mM additional NaCl (i.e., with ∼7% increase in medium Na+ from 145 to 155 mM) (Fig. 2). However, 10 mM of additional KCl (i.e., an increase of [K+ ] of more than three-fold, from 4.4 to 14.4 mM) did reduce the Y1 internalization by 12% (Fig. 2). Internalization of [125 I]transferrin was also inhibited by Li+ in a concentration-dependent fashion, and by nearly 50% at 10 mM (Fig. 2). The surface binding of [125 I]transferrin was elevated in the presence of Li+ in a concentrationdependent fashion (the inset of Fig. 1B). Addition of 10 mM

Fig. 2. A comparison of effects of added cations on the internalization of Y1selective ligand LP-PYY and of human transferrin in CHO cells expressing the cloned guinea pig Y1 receptor. The cells were cultured with indicated concentrations of added cations for 24 h before incubation for 20 min with [125 I]-labeled LP-PYY or transferrin. Data are averages of six measurements, shown with the respective standard errors. Asterisks indicate significance in Student’s t-test vs. no Li+ (* p < 0.05; ** p < 0.01).

M.S. Parker et al. / Neuroscience Letters 374 (2005) 43–46

Fig. 3. Effect of increasing agonist concentration on the internalization of Y1 agonist LP-PYY and of transferrin in CHO cells treated with 1–10 mM Li+ . The length of labeling with iodinated agonists was 20 min. The data are averages of three separate experiments, shown with the respective standard errors. Concentrations of the labeled and the non-labeled agonist were summed for plotting. For control (no Li+ ) groups, differences in Dunnett’s t-test with p < 0.05 vs. all Li+ groups are indicated by ampersands (&). Increases with p < 0.05 in the test for [125 I]-labeled agonist uptake by nonlabeled agonists are indicated by asterisks (*). Note that the concentration axis is in base 10 logarithms. (A) Effect of increasing the input of unlabeled LP-PYY in the range of 0.1–10 nM on the internalization of 0.02 nM [125 I]labeled LP-PYY. (B) Effect of increasing the input of unlabeled transferrin in the range of 1–30 nM on the internalization of 1 nM [125 I]-labeled human transferrin.

KCl induced a 14% reduction of transferrin internalization (Fig. 2). The inhibition of LP-PYY internalization by Li+ could be reduced by increasing concentration of the Y1 agonist in the range of 0.1–10 nM (Fig. 3A). A similar decrease of Li+ -induced inhibition was observed at increasing concentrations of non-labeled transferrin in the range of 0.3–30 nM (Fig. 3B). With either agonist, the inhibition by 1 mM Li+ was overcome at higher agonist molarities (1–10 nM for the Y1 agonist, 3–30 nM for transferrin). Internalization without Li+ and at 1 mM Li+ was significantly increased by non-labeled Y1 agonist at up to 0.6 nM (Fig. 3A), and by added transferrin at up to 3 nM (Fig. 3B). At 3 mM Li+ , a significant increase over tracer transferrin was found with 1 nM added non-labeled transferrin (Fig. 3B). Separation of light (primary or early) and late or secondary endosomes was done in Percoll gradients, based on prior studies of Urade et al. [21] and Tjelle et al. [20] and adapted in our previous studies of Y receptor internalization [15,17]. Treatment with 3 and 10 mM Li+ induced, respectively, a significant and a very significant decrease of the labeling of processing (secondary) endosomes found at densities of 1.04–1.06 in Percoll gradients (Fig. 4A). In Li+ -treated cells there also was a decrease in the apparent density of particulates (plasma membranes and primary endosomes) containing the bulk of receptor-attached agonist (Fig. 4A). A smaller but also significant decrease was found at densities of 1.04–1.06 with receptors labeled by [125 I]transferrin (Fig. 4B). The plasma membrane/primary endosome peak of radioactivity was shifted toward a lower

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Fig. 4. Changes induced by Li+ treatment in the Percoll gradient density distribution of endosomal particulates containing internalized Y1 agonist [125 I]LP-PYY or [125 I]transferrin. The cells were exposed to 3 or 10 mM Li+ for 24 h prior to labeling for 20 min with the above tracers. The results are averages of three gradient profiles. The 1.01–1.02 density zone contained primary (early) endosomes and plasma membrane fragments, the 1.04–1.06 zone secondary endosomes, and the 1.10–1.11 zone mainly dense granules and lysosomes. See Ref. [15] for details and references about assignation and characterization of these zones. (A) Distribution of [125 I]LP-PYY. The 1.04–1.06 density zone (secondary endosomes; see Ref. [15]) contained 11.6 ± 0.2% (control), 8.1 ± 0.5% (3 mM Li+ ), and 6.9 ± 0.8% (10 mM Li+ ) of total radioactive material in the gradient. Li+ averages were significantly different from control in Student’s t-test. (B) Distribution of [125 I]transferrin. The 1.04–1.06 density zone (secondary endosomes; see Ref. [15]) contained 10.1 ± 0.2% (control), 8.8 ± 0.4% (3 mM Li+ ), and 6.7 ± 0.38% (10 mM Li+ ) of total radioactivity. Li+ averages were significantly different from control in Student’s t-test.

density at 10 mM, but not at 3 mM of Li+ , for both receptors (Fig. 4). Our results show a tandem inhibition of internalization by Li+ treatment for two receptors known to be largely endocytosed and cycled via the fast recycling endocytotic compartment [5,8,14,17]. At 10 mM Li+ , the inhibition is associated with a decrease in density of light endosomes, which could reflect decreased compaction of primary endosomes. At lower [Li+ ], the inhibition could be alleviated by increase in concentration of agonist peptides, which points to importance of binding site occupancy in counteracting the effect of Li+ , and could be connected to G-protein interactions of both the lithium ion and the liganded receptors. By analogy with known sensitivity to lithium of the ␣subunits of heterotrimeric G-proteins [1,12], targets of Li+ could include GTP-operated endosome processors, such as dynamins [7], the large and aggregating endocytotic processor proteins. Small GTPases participating in various stages of endocytosis, such as Rab [5] and Rho [6] G-proteins, may also be affected by lithium, possibly via Mg2+ -sensitive sites (as would be expected from a modeling study with Gi␣ [12]). A reduced receptor–G-protein coupling could also lead to a lower activity of effectors such as adenylyl cyclases and protein kinases, as noted in a chronic Li+ treatment paradigm [4]. Clearance of Y peptides is significantly related to internalization of Y1 group receptors, especially the fast-cycling Y1 and Y4 receptors [2,8,14,18]. A decrease in cycling might contribute to increased levels of extracellular Y peptides in

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animal models [10,22], which could be of interest in regard to obesity associated with chronic Li+ treatment (e.g. [19]). Other neuropeptide receptors sharing fast cycling with Y1 group receptors, e.g. vasopressin receptors, could be affected by Li+ in a similar way [9]. Effect of Li+ to reduce agonist internalization via the Y1 receptor is not related to changes in agonist peptide attachment to the Y1 binding site, as Li+ concentrations of up to 10 mM do not affect the Y1 binding to particulates from disrupted cells [13]. The observed increase by Li+ treatment of agonist binding to surface transferrin receptors (and to a lesser degree also to surface Y1 receptors) should be connected to the decreased rate of receptor internalization. A lower inhibition by Li+ of the labeling of secondary endosomal complement of transferrin relative to the Y1 receptor may reflect a lower intracellular pool size of the natively expressed transferrin receptor. The CHO cells used also had a low density of surface transferrin receptors, apparently not in excess of 2000 sites/cell (see the inset of Fig. 1B). Transferrin internalization is generally perceived as a model of recycling endocytosis, involving mechanisms that are largely shared by structurally very different rhodopsinlike receptors, including the Y1 receptor (as already indicated in Ref. [17]). The large similarity in patterns of Li+ inhibition of internalization for the two receptors indicates that internalization of many recycling receptors (including a large array of neurotransmitter receptors) could be to some extent affected by Li+ .

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