Regulation of Melatonin 1a Receptor Signaling and Trafficking by Asparagine-124

Cole S. Nelson, Masayuki Ikeda, Heinrich S. Gompf, Mindi L. Robinson, Nadine K. Fuchs, Tohru Yoshioka, Kim A. Neve and Charles N. Allen

Center for Research on Occupational and Environmental Toxicology (C.N., M.I., H.G., M.R., N.F., C.A.), Departments of Physiology and Pharmacology and Behavioral Neuroscience (K.N.), Oregon Health Sciences University, Portland, Oregon 97201; Medical Research Service (K.N.), Veterans Affairs Medical Center, Portland, Oregon 97201; Advanced Research Institute for Science and Engineering (M.I.), Waseda University, Tokyo 169, Japan; and Department of Molecular Neurobiology (T.Y.), School of Human Sciences, Waseda University, Tokorozawa 359, Japan

Address all correspondence and requests for reprints to: Charles N. Allen, CROET L606, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Road, Portland, Oregon 97201. E-mail: allenc{at}ohsu.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Melatonin is a pineal hormone that regulates seasonal reproduction and has been used to treat circadian rhythm disorders. The melatonin 1a receptor is a seven- transmembrane domain receptor that signals predominately via pertussis toxin-sensitive G-proteins. Point mutations were created at residue N124 in cytoplasmic domain II of the receptor and the mutant receptors were expressed in a neurohormonal cell line. The acidic N124D- and E-substituted receptors had high-affinity 125I-melatonin binding and a subcellular localization similar to the neutral N124N wild-type receptor. Melatonin efficacy for the inhibition of cAMP by N124D and E mutations was significantly decreased. N124D and E mutations strongly compromised melatonin efficacy and potency for inhibition of K+-induced intracellular Ca++ fluxes and eliminated control of spontaneous calcium fluxes. However, these substitutions did not appear to affect activation of Kir3 potassium channels. The hydrophobic N124L and N124A or basic N124K mutations failed to bind 125I-melatonin and appeared to aggregate or traffic improperly. N124A and N124K receptors were retained in the Golgi. Therefore, mutants at N124 separated into two sets: the first bound 125I-melatonin with high affinity and trafficked normally, but with reduced inhibitory coupling to adenylyl cyclase and Ca++ channels. The second set lacked melatonin binding and exhibited severe trafficking defects. In summary, asparagine-124 controls melatonin receptor function as evidenced by changes in melatonin binding, control of cAMP levels, and regulation of ion channel activity. Asparagine-124 also has a unique structural effect controlling receptor distribution within the cell.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
MELATONIN IS AN important neurohormonal modulator of circadian rhythms (1, 2). It is rhythmically secreted from the pineal gland with circulating concentrations increased during the night (1). Melatonin administration can shift the phase of circadian rhythms with respect to the daily light/dark cycle (3) and has been used to treat circadian rhythm disorders (2, 4). Two high-affinity receptors for melatonin [1a (mt1, Ref. 5) and 1b (MT2, Ref. 6)], are expressed in the central nervous system of mammals in regions including the neonatal pituitary (7), pars tuberalis (8, 9), retina (6, 10), and the hypothalamic suprachiasmatic nucleus (SCN, Ref. 11), which is the biological seat of the circadian clock (12). The most widely expressed melatonin receptor within the SCN is the 1a subtype (6, 13, 14), and this subtype mediates melatonin suppression of action potential firing frequency (14). Presumably, this action is the result of melatonin receptor modulation of ion channels (15).

The melatonin 1a receptor is a seven-transmembrane domain (7TM) receptor (5) that signals intracellularly via G proteins (16). Melatonin receptors regulate a wide range of pertussis toxin (Ptx)-sensitive intracellular messenger pathways (17, 18, 19). These activities include inhibition of forskolin (Fsk)-stimulated cAMP production (5, 20), potentiation of Gs-stimulated adenylyl cyclase activity (21), long-term sensitization of Fsk-stimulated cAMP production (22), and potentiation of PGF2{alpha}-activated PLC (19). Melatonin receptors also couple to Ptx-insensitive Gz to control cAMP (23) and Gq to control intracellular calcium (24). Coupling to Gq may also mediate activation of PKC (25) by melatonin. In addition to intracellular second messengers, melatonin receptors activate Kir3 (GIRK) inward rectifier potassium channels (15). Kir3 channels are widely expressed in the brain including the SCN (26, 27) and pituitary (26) and are directly activated by membrane-bound ß{gamma}-subunits (28) released by Gi/Go-proteins. In rat pituitary gonadotrophs, melatonin modulates calcium channels (7), which can also be directly inhibited by ß{gamma}- subunits (29). Thus, melatonin receptor regulation of these ion channel types through G proteins is important in understanding the activities of melatonin in different tissues in vivo.

Recently, a G (Gly) to T (Thr) point mutation was identified in TMVI of the human melatonin 1a receptor, which controls melatonin binding (30). This demonstrated that, as for other monoamine receptors, binding of the cognate ligand occurs within the receptor cleft formed by the 7TM domains. However, receptor sequences that control signaling through G proteins have not been identified. Cytoplasmic domain II of 7TM domain receptors lies between TMIII and TMIV. This region has been studied in several biogenic amine and peptide receptors and shown to be involved in signal transduction through G proteins (31). An amino acid sequence motif D/ERY/WXXI/VXXPL has been described near the end of TMIII (32). Mutation of the highly conserved acidic residues (D, Asp or E, Glu) found in virtually all 7TM receptors has caused effects ranging from loss of function (33, 34) to constitutive activity (35, 36, 37, 38, 39). For instance, {alpha}2A-adrenergic receptors mutated from DRY to NRY in this position are unable to inhibit cAMP (33). Because melatonin receptors are unusual in possessing the neutral amino acid N (Asn) in this position, point mutations were created in the human melatonin 1a (mt1) receptor including consensus reversions to D/ERY at position N124. The effects of these mutations with respect to receptor expression, ligand binding and signal transduction to cAMP production, potassium channels, and intracellular calcium fluxes were then examined in a stably transfected neurohormonal cell line. The unusual transition from aspartate (D) to asparagine (N) at this position within the melatonin receptor is not a conservative change but a meaningful divergence from the majority of other 7TM receptors. This amino acid has functional control of cAMP levels and ion channel activity and a unique effect on melatonin binding and receptor distribution within the cell.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cellular Expression of Human mt1 Receptor N124 Point Mutants
An N-terminal peptide tag was fused to the human mt1 receptor using the receptor cDNA (5). Point mutations were then introduced at position N124 in cytoplasmic domain II by oligonucleotide site-directed mutagenesis (Fig. 1AGo). Dissociation constant (Kd) and Bmax values for stably selected cell lines were determined by 125I-melatonin saturation binding and nonlinear regression analysis (Fig. 1BGo and Table 1Go). The average Kd values for the tagged N124N (wild-type) melatonin receptor agreed with reported values (5). N124D and N124E receptors showed slightly increased Kd values compared with wild-type receptor (N124D, P <= 0.025; N124E, P <= 0.029, Table 1Go). For N124N (wt), N124D, and N124E receptors, Bmax values were between 200 and 600 fmol/mg of protein (Table 1Go). 125I-melatonin binding to untransfected AtT20 cells was not detected in three separate assays.



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Figure 1. Stable Expression of Human Melatonin Receptor and N124 Point Mutants in AtT20 Cell Lines

A, Partial amino acid sequence is shown for both human melatonin receptors. N124 is in bold font and point mutations are listed below the sequences. B, 125I-melatonin binding analysis of human mt1 receptors stably expressed in AtT20 cells. Representative saturation binding curves for wild-type and point mutant receptors are shown. Nonspecific binding of 125I-melatonin was determined using 1 µM I-melatonin. Average Kd and Bmax values for 125I-melatonin determined using nonlinear regression analysis are given in Table 1Go. C, Confocal immunofluorescent staining of N-terminal fusion epitope for stably expressed melatonin receptors. Cells were stained with anti-Xpress monoclonal and goat antimouse-A488 antibodies and imaged by confocal microscopy using a 60x objective in 0.5 µm slices (see Materials and Methods).

 

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Table 1. 125I-Melatonin Binding to hMel1a Wild-Type and Mutant Receptors Expressed in AtT20 Cells

 
Receptor expression for each cell line was evaluated by staining with a monoclonal antibody specific to the N-terminal peptide fusion epitope and visualized by confocal imaging (Fig. 1CGo). N124N (wt), N124D, and N124E cell lines showed both internal inclusions and peripheral localization, which suggested the presence of receptor protein in both the internal synthetic and surface membrane compartments (Fig. 1CGo). Similar staining patterns were not found in cells incubated with only secondary antibody or in untransfected cells incubated with both primary and secondary antibodies. Thus, the peptide fusion epitope did not appear to alter binding to the wild-type receptor (N124N), and the point mutations N124D and E had a comparatively minor effect (~2-fold) on the affinity of 125I-melatonin. N124D and E mutations also did not appear to alter the cellular distribution of the receptors.

Inhibition of cAMP Production
Melatonin inhibition of Fsk-stimulated cAMP production was concentration dependent in N124N (wt) expressing cells (IC50 = 0.88 nM; Fig. 2AGo). Melatonin did not inhibit Fsk-stimulated cAMP production in the parent AtT20 cells (2 ± 2% inhibition, data not shown). The concentration dependence for inhibition of cAMP production was affected by N124D (IC50 = 4.4 nM, P = 0.03; Fig. 2BGo) but not N124E (IC50 = 2.2 nM, P = 0.64; Fig. 2CGo) point mutations. Melatonin efficacy was significantly decreased for N124D (27.5 ± 2.4% inhibition at 1 µM, P < 0.001) and N124E (16.6 ± 1.9% inhibition, P < 0.001) compared with wild-type N124N (40.9 ± 1.6% inhibition). Several independent cell lines were tested for melatonin inhibition of Fsk-stimulated cAMP using a single concentration of melatonin (1 µM, Fig. 2DGo). Melatonin inhibition of cAMP production in N124N (wt) cell lines (47 ± 3%, Fig. 2DGo) was significantly greater than in N124D (27 ± 4%, P < 0.002) and N124E (20 ± 4%, P < 0.0003) cell lines. Inhibition of cAMP was not significantly different when comparing N124D and N124E cell lines as groups (P > 0.267). Therefore, the N124D and E receptors were functionally deficient, indicating impaired intracellular signaling by the receptors.



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Figure 2. Inhibition of cAMP Production

Melatonin concentration curves for inhibition of Fsk-stimulated cAMP production are shown for N124N (wt) (panel A), N124D (panel B) and N124E (panel C) cell lines. Error bars indicate SE from three to five separate determinations for each data point. Ptx (0.5 µg/ml) was applied to cell cultures 18–24 h before assay. D, Multiple cell lines expressing N124N (wt), N124D, and N124E receptors were compared for inhibition of cAMP production. Melatonin (1 µM) and somatostatin (1 µM) were applied. Error bars indicate SE for triplicate wells in a single assay. An asterisk (*) indicates reassay of cell line used for the concentration response curves shown in panels A–C.

 
Treatment with Ptx completely blocked inhibition of cAMP by melatonin (Fig. 2Go, A–C) indicating that mutation at N124 affected the primary Gi/Go-protein signaling pathway of the melatonin receptor. AtT20 cells express endogenous somatostatin receptors, which inhibit cAMP production through a similar Gi-protein pathway (40). This was confirmed by somatostatin (1 µM) inhibition of Fsk-stimulated cAMP in untransfected AtT20 cells (63 ± 2% inhibition), which was prevented by Ptx treatment (3 ± 4% inhibition, data not shown). Somatostatin also inhibited Fsk-stimulated cAMP production in each melatonin receptor cell line (Fig. 2Go, A–D) and this inhibition was not significantly different from untransfected AtT20 cells (P > 0.28 for all comparisons, Fig. 2Go, A–C). Therefore, each cell line responded normally to somatostatin and appeared to possess an intact G protein signaling pathway comparable to the untransfected parent cell line.

Potassium Channel Activation
Melatonin receptors have been shown to couple to Kir3.1/3.2 channels in vitro by a Ptx-sensitive mechanism (15). The subunit composition of Kir3 channels in AtT20 cells was determined using RT-PCR on total RNA isolated from the wild-type AtT20 parent cells. PCR products were Southern blotted by probing with subunit specific 32P-oligonucleotides, demonstrating expression of Kir3.1 (GIRK1) and Kir3.2 (GIRK2) subunits but not Kir3.4 (GIRK4) subunits in AtT20 cells (Fig. 3AGo). A similar result was obtained by Northern blot analysis of AtT20 poly-A+ RNA (data not shown). Thus, AtT20 cells express Kir3.1/3.2 channels, the predominant subunit composition found in the brain (27).



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Figure 3. Potassium Channel Activation

A, Molecular identification of Kir3 channels expressed in AtT20 cells. RT-PCR was performed on total RNA from wild-type AtT20 cells, OS3 adrenal cell line (negative control, Ref. 61 ), and mouse forebrain (positive control, Ref. 28 ) using oligonucleotide primers specific to Kir3.1, 3.2, or 3.4 channel subtypes. Southern blots were subsequently performed using 32P-end-labeled oligonucleotides specific to each channel subtype. Positions of mol wt markers are shown to the left of each Southern blot (bp). Ethidium bromide-stained control PCRs are also shown for cyclophilin that were performed using 10-fold diluted cDNAs to evaluate cDNA quality. Cyclophilin controls for 3.1 and 3.2 are shown below the 3.1 blot only. B, Kir3 activation by stably transfected human melatonin receptors. Patch clamp recordings in whole-cell configuration were made from cells voltage clamped near the resting potential at -45 mV and voltage steps applied to -100 mV. C, Dose- dependent activation of potassium currents by melatonin. Current amplitudes ± SE for melatonin responses as shown in panel B (n = 3–10 for each data point). Ptx (0.5 µg/ml) was applied to cells in culture 18–24 h before assay.

 
Melatonin activated K+ currents with similar EC50 values for the N124N (7.6 nM), N124D (9.3 nM), and N124E (2.2 nM) cell lines (Fig. 3Go, B and C). Melatonin application to wild-type AtT20 cells did not induce detectable currents in cells that subsequently responded to somatostatin (data not shown). Ptx blocked responses to melatonin (1 µM, Fig. 3CGo) and somatostatin (100 nM, data not shown) in all cell lines. Thus, unlike inhibition of cAMP production, mutation to N124D or N124E did not appear to have a functional effect on Ptx-sensitive coupling to endogenous Kir3 G protein-activated K+ channels in AtT20 cells.

Inhibition of Intracellular Ca++ Fluxes
Melatonin receptors inhibit voltage-dependent calcium channels in vivo (41). AtT20 cells express several voltage-dependent calcium channel types including L-, N- and P/Q-channels (42). These channels can be activated by depolarization of the cell membrane with high external K+ (HiK, 30 mM; Ref. 29). HiK application to AtT20 cells induced an increase in intracellular calcium measured by Fura-2 fluorescence ratio imaging that was approximately 2-fold over background (Fig. 4Go). Repeated application of HiK to untransfected parent AtT20 cells caused a basal, nonspecific reduction of K+-induced intracellular Ca++ fluxes (13 ± 4% inhibition, data not shown). For the N124N (wt) cell line, melatonin caused a reversible, dose-dependent inhibition of K+-induced calcium influx with an EC50 = 1.2 nM (N124N = 46 ± 3% inhibition at 100 nM; Fig. 4Go, A and B). The N124D and N124E mutations strongly compromised melatonin efficacy and potency for inhibition of K+-induced intracellular Ca++ fluxes (N124D = 23 ± 2% and N124E = 24 ± 3% inhibition at 100 nM; Fig. 4BGo). This appeared to be specific to the melatonin receptors because somatostatin (1 µM) applied to the same cell lines reversibly inhibited K+-induced calcium fluxes (N124D = 48 ± 5%, N124E = 37 ± 7%; data not shown) similar to the wild-type receptor (N124N = 31 ± 4%, data not shown). Therefore, point mutation of N124 to either D or E interfered strongly with signal transduction to voltage-dependent calcium channels.



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Figure 4. Inhibition of Intracellular Ca++ Fluxes

A, Time-based plot for inhibition of K+-induced intracellular calcium flux by melatonin. Bars indicate perfusion of HiK for 30 sec at 7-min intervals and melatonin (100 nM) for 90 sec during the second HiK stimulation. Letters correspond to time points as follows: a, baseline; b, first 30 mM BSS (HiK) stimulation; c, combined melatonin and HiK; and d, recovery HiK stimulation. Images were recorded with a 40x objective. B, Dose- dependent inhibition of K+-induced intracellular Ca++ fluxes by melatonin. The dose response for melatonin inhibition of K+-induced intracellular Ca++ fluxes is expressed as the ratio of peak response after melatonin/HiK application "c" divided by peak response after HiK application "b." Data points shown are mean ± SE with n = 32–145 total cells measured during three to five separate experiments. Ptx was applied to the cells overnight at a concentration of 500 ng/ml. C, Inhibition of spontaneous Ca++ oscillations. Representative trace showing melatonin inhibition of spontaneous Ca++ oscillations in the N124N (wt) cell line. Bars indicate application of 100 nM melatonin for 90 sec at the concentration shown. D, Point mutation at N124 to D or E and Ptx treatment decouples melatonin inhibition of spontaneous Ca++ oscillations. Bars represent percentage of population exhibiting spontaneous Ca++ oscillations that were inhibited by melatonin application. Individual cells were recorded from groups in four to six separate experiments. Ptx was applied to the cells overnight (~18 h) at a concentration of 500 ng/ml.

 
Whereas inhibition of cAMP or activation of K+ channels (Fig. 2Go and 3Go, respectively) was completely eliminated by Ptx treatment, melatonin inhibition of K+-induced intracellular calcium fluxes was only partially sensitive to Ptx (N124N = 23 ± 1% inhibition at 100 nM with Ptx, Fig. 4BGo). This suggested that melatonin receptor inhibition of Ca++ channels may occur through both a major Ptx-sensitive and a minor Ptx-insensitive pathway.

AtT20 cells also showed spontaneous calcium oscillations, which have been shown to correlate with the firing of spontaneous action potentials (43). Spontaneous intracellular calcium oscillations recorded by Fura2 ratio imaging were found in a subset of cells from each line (N124N, 48 ± 12%; N124D, 45 ± 12%; N124E, 35 ± 7% spontaneously active cells) and the parent AtT20 cell line (51 ± 13% spontaneously active cells). The predominant response to melatonin in spontaneously active N124N (wt) cells was a reversible inhibition that was sensitive to Ptx treatment (Fig. 4Go, C and D). Melatonin did not inhibit spontaneous intracellular calcium fluxes in untransfected AtT20 cells (data not shown). Inhibition of spontaneous calcium oscillations by melatonin was significantly reduced in N124D and N124E cell lines (Fig. 4DGo). Therefore, wild-type N124N melatonin receptors inhibit calcium oscillations in spontaneously active AtT20 cells by a Gi/Go-mediated mechanism. Mutation of N124 to either D or E appeared to eliminate the melatonin control of spontaneous calcium fluxes.

Golgi Retention of Nonfunctional Mutants
Cell lines expressing N124A, -L, and -K receptors were chosen by intensity of staining for the N-terminal peptide tag but showed no significant binding of 125I-melatonin (Fig. 1Go and Table 1Go). In addition, N124A and N124K receptors showed a different staining pattern compared with N124N (wt) cells: a single large intracellular inclusion and a decreased staining intensity near the periphery of the cells (Fig. 1CGo). N124L cell lines showed staining near the surface membrane but with a more aggregated appearance than for N124N (wt), N124D, and N124E receptors (Fig. 1CGo). In those N124A and N124K cells that stained for {alpha}-mannosidase II, receptor staining showed a strong colocalization (Fig. 5AGo), indicating retention of the receptor proteins in the Golgi (44). Receptor staining was also observed which did not colocalize with {alpha}-mannosidase II, indicating presence of the additional receptors in a separate compartment (Fig. 5BGo). Thus, amino acid position N124 had far-reaching effects on the structure and conformation of the melatonin receptor. In addition to functional effects on melatonin potency and efficacy seen with N124D and N124E mutations, other substitutions at this position caused a loss of agonist binding and retention of the receptor in the Golgi. This indicates that N124 controls not only receptor signaling function but is critical for correct receptor structure.



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Figure 5. Nonfunctional N124A and K Point Mutants Are Retained in the Golgi

A, Immunofluorescent staining of the Golgi-specific enzyme {alpha}-mannosidase II ({alpha}ManII), staining of the N-terminal fusion epitope attached to the N124A and K receptors (Receptor) and their colocalization (Overlay). B, Enlargement of selected area from field shown in panel A. Overlay: cells were stained with rabbit anti-{alpha}-mannosidase II and goat antirabbit-A546 antibodies and anti-Xpress monoclonal and goat antimouse-A488 antibodies and imaged by conventional immunofluorescent microscopy using a 63x objective.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Melatonin receptors are unusual in possessing the neutral amino acid N (Asn) at position N124 in the second cytoplasmic domain instead of the acidic D (Asp) or E (Glu) residues found in other 7TM domain receptors. Point mutations were created in this position of the human mt1 receptor, and the receptors were stably expressed in a neurohormonal cell line. The effects of consensus reversions to D or E and mutation to basic K (Lys) or nonpolar A (Ala) and L (Leu) were then examined. Mutants at N124 could be separated into two sets. The first set consisted of mutants that had normal trafficking to the membrane and binding of I-melatonin, but exhibited altered signaling to intracellular effectors. The second set exhibited a loss of melatonin binding combined with aberrant receptor trafficking including retention in the Golgi. Thus, melatonin receptor position N124 controls not only receptor function but also receptor structure as indicated by the observed agonist binding and trafficking defects.

Within the D/ERY motif, the second position R (Arg) residue has been modeled to rotate in and out of the agonist binding cleft and serve as an agonist-sensitive conformational switch controlling G protein interaction (36, 45). This portion of the receptor is proximal to the cell membrane and therefore at an interface between hydrophilic and hydrophobic regions. Thus, the charge character of the residue adjoining the conserved R residue in the D/N position could also influence receptor movement or interaction with G proteins. Substitution of the neutral N (Asn) with nonpolar, hydrophobic L (Leu), A (Ala), or positively charged K (Lys) eliminated agonist binding (Table 1Go) and affected the subcellular distribution of receptor protein (Fig. 1Go). N124A and K receptors showed a staining pattern with a single large intracellular inclusion associated with the medial or trans-Golgi (Fig. 5Go and Ref. 44) and a decreased staining intensity near the periphery of the cells. The same characteristics that endow functional control of signal transduction on this position through flexibility in the receptor may also restrict the allowable amino acids because of structural constraints. Thus, the apparent flexibility of this part of the receptor may have consequences for assembly or folding of the protein and explain the almost absolute conservation of the acidic D/E amino acid residues found in most 7TM receptors.

The N124L mutant also lacked agonist binding (Table 1Go) but had a unique staining pattern near the surface membrane (Fig. 1Go) and thus trafficked out of the Golgi compartments, unlike the N124A and K mutants. But, when viewed through consecutive confocal slices, N124L had a more aggregated appearance than N124N (wt), D, or E receptors (data not shown). Thus, the N124L mutation may separate a conformational defect in agonist binding from a defect related to trafficking. It is possible that the leucine side chain, which is similar in size and structure to asparagine, caused a less severe disruption of the receptor. These speculations as to the folding and structural defects for N124 mutants may be testable by associational rescue using wild-type receptor or compensatory mutations in cotrafficking molecules to restore agonist binding and cellular trafficking.

Receptor folding and transport can require association with cyclophilins or other chaperonins as for rhodopsin (46) and red/green opsins (47). Misfolded receptors can aggregate and fail to traffic. Alternatively, the point mutations may have interfered with an associated subunit required for folding and trafficking. Odorant receptors have type-specific trafficking chaperones that associate with the nascent receptor in the endoplasmic reticulum (48), and these receptors fail to express in some cell lines, presumably due to the lack of expression of their required trafficking chaperone. For melatonin receptors, this seems unlikely as a cell line-specific failure of ectopic expression (49) has not yet been reported. LH receptor mutated at the homologous position, E441Q, was retained in a detergent-soluble compartment that could not be detected with a live cell binding assay (50), indicating retention or altered internalization of the mutant receptor. This is of interest as the amino acid glutamine (Q) is of similar structure and neutral charge as asparagine (N). Thus, an analogous, but inverse transition may have resulted in a similar phenotype; however, intracellular localization of the mutant receptor by fluorescent staining or a similar method was not examined.

Identification of the functional determinants that control second messenger signaling is important for understanding the actions of melatonin in different tissues. Melatonin receptors signal to a broad range of intracellular effectors. Acidic substitutions for N124 had distinct effects on each of the three signaling pathways that were assessed. The maximal inhibition of cAMP production by melatonin was decreased but still detectable (Fig. 2Go), whereas Ptx-sensitive inhibition of calcium influx was abolished (Fig. 4Go) and activation of Kir3 potassium channels was unchanged (Fig. 3Go). These results could be explained by a mutation-induced, selective loss of coupling efficiency by the receptors for particular combinations of G protein {alpha}ß{gamma}-subunits. The functional outcome of this selective loss of coupling would then depend on the G protein specificity of the effector. For example, muscarinic and somatostatin receptors have been shown to prefer specific {alpha}ß{gamma} combinations in coupling to calcium channels (57). In addition, N-type calcium channels are selective for ß-subunits, binding strongly to ß1 and ß2 and weakly to ß3, ß4, and ß5 (51). A selective loss of coupling by N124D and N124E receptors to Ptx-sensitive Gß1- and Gß2-containing heterotrimers might result in the observed complete loss of Ptx-sensitive and sparing of Ptx-insensitive inhibition of calcium channels. On the other hand, Kir3 (GIRK) potassium channels have no specificity for subtypes, except for not responding to ß5 (52). Thus, activation of Kir3 channels would not be affected by a mutation-induced loss of coupling to Gß1- and Gß2-containing heterotrimers. Alternatively, the relatively small K+ currents evoked in AtT20 cells may have limited the ability to resolve differences in signaling strength. Adenylyl cyclases are not known to show a preference for inhibition by particular subtypes of G{alpha}i or {gamma} (53), so partial loss of inhibition of adenylyl cyclase is consistent with a selective loss of mutant receptor coupling to a subset of the total pool of heterotrimers that are activated by the wild-type receptor.

Although all three pathways were sensitive to inhibition by Ptx treatment, indicating the involvement of the Ptx-sensitive G proteins Gi and Go, there was a Ptx-insensitive component of the regulation of K+-activated calcium fluxes by the melatonin receptor (Fig. 4Go). Similarly, {alpha}2-adrenergic receptors have been shown to inhibit calcium channels by both Ptx-sensitive and -insensitive G proteins and, as observed for melatonin receptors, the IC50 for agonist was significantly higher for Ptx-insensitive inhibition (54). This could reflect coupling to another G protein pathway. Indeed, a secondary molecular interaction between melatonin 1a receptors and Gq has recently been demonstrated (24).

For 7TM receptors signaling to phospholipases through Gq proteins, the transition from D to N at a position homologous to N124 appears to be an essentially conservative substitution. {alpha}1B-adrenergic receptor mutation at D142 resulted in constitutive inositol phosphate accumulation for most of the substitutions tested (36). However, D142N and E showed no major effect on agonist affinity and only small effects on constitutive inositol phosphate accumulation (35, 36). A similar result was found for the Gq-coupled m1 muscarinic (D122N, Ref. 37), m5 muscarinic (D127N, Ref. 38), and 5HT2 (D172N, Ref. 39) receptors where the homologous, inverse mutations had little effect on agonist-stimulated or constitutive activities. Thus, it is reasonable to expect a disruption of Gi but not Gq signaling by a mutation at N124 in melatonin receptors, but this remains to be tested.

The "D/ERY" motif found in virtually all other 7TM receptors has been shown to affect signal transduction through Gi, Gq, and Gs depending on the receptor examined. The effect of this type of substitution in "D/ERY" receptors or other receptors possessing the neutral amino acid N (Asn), which includes the platelet-activating factor receptor (55), tagged so that their trafficking can be examined remains to be tested. Whether due to the physical requirements of melatonin as a ligand, the required G protein specificity for proper melatonin signaling, or another structural requirement related to receptor synthesis, folding, or transport, position N124 clearly differentiates the melatonin receptor from most 7TM receptors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Construction and Mutagenesis
The plasmid pcDNA6.1 was constructed by ligation of the ScaI/XhoI fragments of pcDNA3.1A (Invitrogen, San Diego, CA; fragment containing PCMV, T7, start codon, Xpress epitope, polyhistidine, and multiple cloning site) and pcDNA6A (Invitrogen; fragment containing multiple cloning site, bovine GH polyA, blasticidin, and origin of replication) to create a vector with N-terminal peptide epitope and His tags, and blasticidin antibiotic resistance. The human mt1 receptor was kindly provided by Dr. Steven Reppert (GenBank accession no. U14109, Ref. 5). The human mt1 receptor cDNA was excised from pcDNA3 using HindIII and XbaI, ligated into pSK-, and then excised with XhoI and XbaI and subcloned into pcDNA3.1HisC to create hMel1a/pcDNA3.1HisC. hMel1a/pcDNA6.1 was then constructed by the same method as pcDNA6.1 (described above) using ScaI/XbaI fragments of hMel1a/pcDNA3.1HisC and pcDNA6A.

Point mutations were generated using oligonucleotide-directed mutagenesis as described (Transformer Site-Directed Mutagenesis, CLONTECH Laboratories, Inc. Palo Alto, CA) using the following oligonucleotides (Life Technologies, Inc., Gaithersburg, MD): 5'-GTG ACT GGT GAA TAC TCA ACC AAG-3' (selection marker), 5'-GGC ATC GCC ATC GAC CGC TAC TGC-3' (N124D), 5'-GGC ATC GCC ATC GAG CGC TAC TGC-3' (N124E), 5'-GGC ATC GCC ATC GCC CGC TAC TGC-3' (N124A), 5'-GGC ATC GCC ATC CTC CGC TAC TGC-3' (N124L), 5'-GGC ATC GCC ATC AAG CGC TAC TGC-3' (N124K). Each cDNA was then completely sequenced by automated sequencing (Stretch 373, Perkin-Elmer Cetus, Norwalk, CT) and compared with the published receptor sequence using the Factura/Autoassembler (Perkin-Elmer Cetus) and GCG suite of programs (Accelrys, Inc., San Diego, CA) to confirm the presence of a single, exclusive mutation site.

RT-PCR
RT-PCR was performed by extraction of total RNA from culture cells or brain tissue prepared by a modified guanidinium thiocyanate extraction (56). RNA (1–5 µg) was primed using oligo-d(T)18 and reverse transcribed with Superscript-RT (Life Technologies, Inc.) under standard reaction conditions and used as template for PCRs performed using Taq polymerase (Promega Corp., Madison, WI) in a Perkin-Elmer Cetus 9600 PCR machine in standard reaction buffer. Sequences of the oligonucleotide used for Kir3 channel RT-PCR and Southern blot were: 3.1–5', 5'-AACAGCCACATGGTCTC-3'; 3.1–3', 5'-GTGCTAATGTCATCTAGTC-3'; 3.1-probe, 5'-TTGACCAACTTGAACTGGATGTAGGTTTTAGTACAGG-3'; 3.2–5', 5'-TCAACGCCTTCATGGTA-3'; 3.2–3', 5'-CTTGATCCACACTAGGA-3'; 3.2-probe, 5'-CTCCAAAGCGCAGCTGCCTAAAGAGGAACTGGAGA-3'; 3.4–5', 5'-GTATGGCTTCAGAGTCATT-3'; 3.4–3', 5'-TTCATCCTTCTCGGCCT-3'; 3.4-probe, 5'-CTTTTGTGGAGATGTCTCGTGCTCAACTGGAACAG-3'; cyclophilin-5', 5'-CTGGTCTTGCCATTCCTGGACCCA-3'; cyclophilin-3', 5'-CATGTGCCAGGGTGGTGACTTCAC-3'. The conditions for Kir3 PCR cycling reactions were: 94 C/30 sec; 58 C/30 sec; 72 C/60 sec for 35 cycles. The conditions for cyclophilin PCR cycling reactions were: 94 C/60 sec; 60 C/30 sec for 35 cycles.

Cell Culture and Transfection
Mouse anterior pituitary tumor cells (AtT-20/D16–8) cells are grown in DMEM (4,500 mg/liter glucose, Life Technologies, Inc.) plus 10% FBS (Life Technologies, Inc.) at 37 C and 5% CO2. For stable transfections cells were trypsinized and plated (5 x 104 cells/ml, 0.3 ml/cm2) and at 1 d after plating were transfected using Lipofectamine Plus (Life Technologies, Inc., 0.1 ml/cm2, 1 µg/ml plasmid, 3 µl/ml Lipofectamine Plus, 1.5 µl/ml lipofectamine) for 4–18 h before media change with media changes every 24 h thereafter. Cells were selected using blasticidin S.HCl (2.5 µg/ml, Invitrogen) starting at 2 d after transfection, reapplied with media daily for 10 d and every 2 d subsequently. At 10–14 d after transfection, cells were scraped and plated at high dilution and single colonies were picked to establish cell lines. For routine maintenance, blasticidin and media were renewed every 4 d. For functional assays blasticidin and media were changed every 24–48 h and always applied the day before assay.

125I-Melatonin Binding
AtT20 cells were harvested by scraping in PBS, pH 7.4, pelleted, and resuspended in approximately 10 ml lysis buffer at 4 C containing (mM): 50 Tris-HCl, 1 PMSF, and 1 EGTA at pH 8 and homogenized (Brinkmann Instruments, Inc., Westbury, NY). Membranes were then pelleted at 36,000 x g for 20 min and resuspended in sodium ion-free assay buffer at 25 C containing 50 mM Tris-HCl and 5 mM MgCl2 at pH 7.4. 2-[125I]-melatonin was incubated with membranes (~30 µg total protein/500 µl) in the absence and presence of melatonin for 150 min at 25 C with continuous shaking. The reactions were terminated by filtering over GF/B filters (Whatman, Clifton, NJ) using a 96-well harvester (Tomtec, Orange, CT) at 4 C and washed with pH 7.4 buffer at 4 C containing 10 mM Tris-HCl, 150 mM NaCl. The filters were dried, and each sample was saturated with 50 µl of scintillation fluid and counted using a BetaPlate scintillation counter (Wallac, Inc., Gaithersburg, MD). Data for saturation and competition binding were analyzed by nonlinear regression using commercial software (Kaleidagraph) according to the formula: y = Bmax * x/(x + Kd) where y = concentration of bound 125I-melatonin and x = concentration of free 125I-melatonin.

Immunofluorescent Imaging
AtT20 cells lines were trypsinized and plated in four-well permanox plastic chamber slides (Nalge Nunc International, Rochester, NY) for 2–4 d with daily media and antibiotic replacement. Cells were fixed in 3% paraformaldehyde in PBS (0.58 M Na2HPO4, 0.17 M NaH2PO4, pH 7.4; 0.68 M NaCl) at 4 C for 30 min, washed three times with TBS-GS (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5.5% vol/vol goat serum; Life Technologies, Inc.) for 5 min and preblocked with TBS-GS for 1 h at room temperature. Cells were then incubated overnight with primary antibody (anti-Xpress monoclonal IgG1, Invitrogen, 1:100 dilution; rabbit anti-{alpha}-mannosidase-II serum, Complex Carbohydrate Research Center, University of Georgia, 1:500 dilution) in TBS-GS and washed three times with TBS-GS. Cells were then incubated for 1 h with secondary antibody (goat antimouse IgG-Alexa 488, goat antirabbit Alexa 568; Molecular Probes, Inc., Eugene, OR) at a 1:800 dilution and washed three times with TBS-GS. Chambers were then removed, slides dipped briefly in distilled water, cover slips mounted with Elvanol [120 mM Tris-HCl, pH 8.5; 140 mM 1, 4-diazobicyclo-[2.2.2]-octane; 12% (wt/vol) polyvinylalcohol; 50% (vol/vol) glycerol] and sealed with nail polish.

Confocal imaging was performed at the Oregon Hearing Research Center with an MRC 1024 ES laser scanning confocal imaging system (Bio-Rad Laboratories, Inc., Hercules, CA) with krypton/argon laser excitation at 488 nm attached to an inverted Eclipse TE300 microscope (Nikon, Melville, NY). Images were acquired and processed using LaserSharp (Bio-Rad Laboratories, Inc.) and Photoshop (Adobe Systems, San Jose, CA) software packages. Conventional immunofluorescent images were acquired using a DMIRBE microscope equipped with high numerical oil lenses (63 x 1.32 N.A.; Leica Corp., Deerfield, IL), green fluorescent protein/ENDOW and rhodamine N3 filter sets (Chroma Technology, Brattleboro, VT), a Micromax camera with Sony chip (1300 x 100 pixels; Princeton Instruments, Monmouth Junction, NJ) and Metamorph ver. 4.1 software (Universal Imaging Corp., Westchester, PA).

cAMP Production
For cAMP assay, cells were trypsinized, plated onto 24-well plates (Falcon, Franklin Lakes, NJ), and changed to fresh culture media with antibiotic every 48 h thereafter. At three to five days after passage, 80–90% confluent cells were rinsed three times in DMEM, incubated with 1 mM 3-isobutyl-1-methylxanthine (RBI, Natick, MA), 500 µM 4-(3-butoxy-4-methoxy-benzyl)imidazolidin-2-one (Sigma, St. Louis, MO), and agonist for 10 min before the addition of 100 µM Fsk. After an additional incubation of 20 min, the medium was aspirated, 0.5 ml of 4 mM EDTA, 10 mM Tris-HCl, pH 7.5, were added on ice, and the plates were frozen at -80 C before assay. For assay each sample was harvested, sonicated, boiled for 5 min, placed on ice, and then centrifuged (12,800 x g for 20 min). Each supernatant (50 µl) was assayed for cAMP levels in duplicate according to standard methods (3H-cAMP assay no. TRK432, Amersham Pharmacia Biotech, Arlington Heights, IL). Fsk stimulation typically resulted in 10- to 12-fold cAMP produced over background levels (~4 pmol cAMP per well) in the presence of 3-isobutyl-1-methylxanthine/4-(3-butoxy-4-methoxy-benzyl)imidazolidin-2-one. Results were expressed as a percentage of Fsk-stimulated maximum except for responses using Ptx-treated cells. Ptx treatment caused a nonspecific increase in Fsk-stimulated cAMP levels in all wild-type and melatonin receptor stable cell lines. For Ptx treatment, results were calculated as a percentage of a combined maximum stimulation by both Fsk and Ptx. Curve fits were determined using the logistic growth model (57), which has the functional form: f(x | m0, m1, m2) = y = m0 + m1/(1 + m2e-[x]) where m0 and m1 are minimum and maximum response values calculated for individual response values y at each agonist concentration [x] using nonlinear least squares algorithm provided in the R package (58). Degrees of freedom for the statistical test were calculated by Satterthwaite approximation (59). SE values for the estimated IC50 and EC50 values were found using the delta method (59, 60).

Electrophysiology
For electrophysiological recording the cells were trypsinized, plated at a density of approximately 5 x 104 cells/ml onto washed 18-mm glass coverslips (no. 12–545-84, Fisher Scientific, Pittsburgh, PA) and changed to fresh culture media with antibiotic each day thereafter. Recordings were performed at 3–4 d by placing a coverslip fragment into a recording chamber located on the stage of an inverted microscope equipped with phase-contrast optics. The chamber was perfused at approximately 1 ml/min with extracellular solution, at pH 7.3 and 320 mOsm containing in mM: NaCl, 128; KCl, 30; CaCl2, 3; MgCl2, 2; glucose, 10; and HEPES, 10. Microelectrodes, pulled in two stages (Narishige puller) from thin-walled borosilicate glass (World Precision Instruments, Sarasota, FL), had resistances of 5–7 M{Omega} when filled with a (intracellular) solution, at pH 7.3 and 310 mOsm, consisting of in mM: K+ gluconate, 84; KCl, 38; KOH, 33; CaCl2, 1; HEPES, 10; EGTA, 10; ATP, 3; and GTP, 0.3. Test agents were applied from a U-tube system, while antagonists were applied both through the U-tube and by bath perfusion. Compounds were made fresh before each experiment. Currents were recorded using the whole-cell configuration of the patch-clamp technique and voltage-clamped using an EPC-7 (Adams List), filtered at 2 kHz (-3 dB) with an eight-pole Bessel filter and digitized on-line using an ITC16 A/D interface (Instrutech, Port Washington, NY) and Pulse software (HEKA, Lambrecht, Germany). Curve fits and values for IC50 and EC50 values were determined by nonlinear regression using commercial software (Kaleidagraph 3.0.4) according to the logistic formula: y = c/[1 + exp(H * ln([C50]/[x]))] + b where b and c are minimum and maximum response values, H is a Hill coefficient, and [C50] is the 50% concentration calculated by nonlinear regression for individual response values y at each agonist concentration [x].

Calcium Imaging
AtT20 cell lines were trypsinized and plated in uncoated glass bottom petri dishes (MatTek Corp., Ashland, MA) at a density of approximately 5 x 104 cells/ml, media and blasticidin were changed each day, and cells were assayed at 3 to 4 d. The cells were gently rinsed with buffered salt solution (BSS) consisting of (mM) 128 NaCl, 5 KCl, 2.7 CaCl2, 1.2 MgCl2, 1 Na2HPO4, 10 D-glucose, and 10 HEPES-NaOH of pH 7.4 and then incubated in BSS supplemented with 5 µM fura-2 AM (Molecular Probes, Inc.) and 0.1% albumin for 30 min at 37 C. The cells were rinsed in BSS, incubated at 37 C for 15 min, and then continuously perfused at a flow rate of 3 ml/min.

Fluorescent images were recorded using an upright microscope (Axioskop FS,Carl Zeiss, Thornwood, NY) with a water immersion objective (FL40x/0.8, Olympus Corp., Lake Success, NY). Paired pulse UV excitation (wavelength = 340 nm and 380 nm, each pulse length = 147 msec) was given by a monochromator (Polychrome 2, Till Photonics, Martinsried, Germany) at 6-sec intervals and reflected by a dichroic mirror (FT395 nm, Carl Zeiss). The fluorescent image was band passed (BP500–530 nm, Carl Zeiss), amplified by an image intensifier (C7039–02, Hamamatsu Photonics, Bridgewater, NJ), and exposed to a multiple format cooled charge-coupled device camera (C4880, Hamamatsu Photonics). The UV light exposure, charge-coupled device camera, image sampling, and acquisition were controlled with a digital imaging system (ARGUS HiSCA, Hamamatsu Photonics) installed in a personal computer. All measurements were corrected for background fluorescence. Changes in intracellular concentration of Ca2+ were estimated from the ratio of fura-2 emission intensities. Curve fits and values for IC50 and EC50 values were calculated as for K+ channel measurements shown above.

Statistics
Statistical differences were calculated by unpaired Student’s t test performed with commercial software (Statview 5.0.1, SAS Institute, Cary, NC). Values were considered significantly different when P < 0.05.


    ACKNOWLEDGMENTS
 
We thank Jennifer L. Marino for her expertise and support. We also thank Michael Lasarev for statistical analysis and advice.


    FOOTNOTES
 
This work was supported by NIH Grants NS-036607 and HL-07890 (T35), by the "Research for the Future" program 96L00310 from the Japan Society for the Promotion of Science, by the Ono Pharmaceutical Co., Ltd., and by Veterans Affairs Merit Review.

Abbreviations: BSS, buffered salt solution; Fsk, forskolin; HiK, high external K+; mt1, melatonin 1a receptor; Ptx, pertussis toxin; SCN, suprachiasmatic nucleus; TBS-GS, 50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5.5% vol/vol goat serum; 7TM, seven-transmembrane.

Received for publication September 13, 2000. Accepted for publication May 1, 2001.


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