- Email: [email protected]
Desensitization of melanotropin receptors in COS-7 cells
PII SOO24-3205(96)00083-S
ELSEVIER
DESENSITIZATION
OF MELANOTROPIN
Life Science, Vol. 58, No. 15, pp. 1223-1229,19% Copyright0 1996 Elcevier Science Inc. Printed in the USA All rights ~cwed am-320.5/% sl5.00 + .oo
RECEPTORS
IN COS-7 CELLS’
Willma E. Polgar*, Catherine M. Bitler#, and Lawrence Toll* *Department of Neuroscience SRI International 333 Ravenswood Avenue Menlo Park CA 94025 #Neurex Corporation 3760 Haven Avenue, Menlo Park, CA 94025
(Received
in final form February 5, 19%)
Summary Non-transfected COS-7 cells have been found to possess functional melanotropin receptors on their cell surface. These receptors, and the properties of the melanocyte stimulating hormone (MSH) peptides can be characterized by measuring melanotropin stimulation of CAMP accumulation in the cells. In these cells we studied the ultra-long lasting super agonist [Nle4-D-Phe7]-cx-MSH (NDP-aMSH), and compared it with the endogenous MSH peptides with respect to potency, maximal activity, duration of action, and rate of desensitization. Surprisingly, NDPa-MSH did not act as a full agonist in COS-7 cells. In multiple experiments, it could stimulate CAMP accumulation to approximately 50% of the level of a-MSH, P-MSH and adrenocorticotropic hormone (ACTH). The MSH receptor mediating this activity is unknown. The time course of CAMP accumulation, and the duration of receptor activation was also investigated. In contrast to other systems, NDP-a-MSH did not induce prolonged activity, with respect to CAMP accumulation, in COS-7 cells. The MSH receptors present in COS-7 were found to desensitize rapidly subsequent to pretreatment by any of the MSH peptides. As expected for a partial agonist, the activity of NDP-a-MSH desensitized more rapidly than any of the full agonists. Surprisingly, desensitization induced by pretreatment with NDP-a-MSH also occurred more rapidly than desensitization induced by the other MSH analogs. Key Words: melanocyte stimulating hormone, melanotropin receptors, COW cells a-Melanotropin (cc-melanocyte stimulating hormone, a-MSH) and the other proopiomelanocortin (POMC)-derived peptides have been shown to have a variety of functions, ranging from well known effects on melanocytes and adrenal cortical cells, to more poorly understood central nervous system actions on consciousness, perception of pain, motivation and behavior (I -3). MSH has also been shown to effect natriuresis (4). POMC, or the mRNA encoding it, have been found in a variety of tissues including brain, lung, liver, adrenal, kidney, among others (5). A significant number of structure activity studies have been carried out on MSH and analogs, identifying structural requirements for binding to MSH and adrenocorticotropic hormone (ACTH) receptors (6.7). One of the most significant discoveries of the structure activity studies was the identification of a class of MSH analogs that display extremely high potency, and remarkably prolonged activity. The prototypical long-acting MSH analog is [Nle4-D-Phe7]-a-MSH (NDP-aMSH). This compound has been shown to have prolonged melanotropic activity in lizard and frog Corresponding Author: Lawrence Toll, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025. USA. Telephone, (4 15) 859-380 1; FAX, (4 15) 859-4159; e-mail, toll @unix.sri.com. This work was supported by NIDA grant DA06682 to LT.
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skin in vitro and in viva (8,9). It also produces prolonged stimulation of tyrosinase activity in S91 melanoma cells. Interestingly, this compound induces tyrosinase activity after a very short exposure to the melanoma cells, and its stimulating activity is maintained after removal of the compound from the cells (10). There are other ways to measure activity of MSH and analogs. Receptor stimulation has been shown to mediate an increase in CAMP levels in S91 cells (11,12). Recently, five distinct melanotropin receptors have been cloned and transfected into a variety of cell lines (13-18). As expected, the melanotropins stimulate CAMP accumulation in cells transfected with any of the five melanotropin receptors (MCRs). More recently, we have demonstrated the presence of MCRs in non-transfected COS-7 cells. MSH, ACTH and various analogs mediate an increase in CAMP levels in this cell line, and mRNA encoding MC2R and MC3R have been demonstrated by Northern analysis and polymerase chain reaction (PCR)/Southern analysis (Bitler et al., submitted) In this study we have investigated the time course of the stimulation of CAMP accumulation, and the rate at which MSH and analogs induce desensitization of the CAMP-stimulating response of the melanotropin receptors found in COS-7 cells. We have found that MSH and analogs induce rapid desensitization of MCRs in COS-7 cells, and at least with respect to CAMP accumulation, that NDP-a-MSH is not a superagonist with prolonged activity in these cells. Methods Cells Culture COS-7 cells were grown on 100 mm culture dishes in Dulbeco’s Modified Eagle’s Medium (DMEM) supplemented with 5% fetal bovine serum and 10% horse serum. CAMP Assav COS-7 cells were plated on 6-well plates and used at confluence. Wells were washed once with incubation buffer containing: 130 mM NaCl, 4.8 mM KCI, 1.2 mM KH2P04, 1.3 mM CaCl2, 1.2 mM MgS04, 10 mM glucose, 50 mM isobutylmethylxanthine (IBMX) and 25 mM HEPES, pH 7.4. To each well was added 1 ml buffer, and cells were pre-incubated for 10 min at room temperature. This buffer was aspirated, and replaced with 1 ml fresh buffer containing the appropriate MSH peptide. To determine the time course of activity, cells were then incubated in triplicate for times ranging from 1 to 60 min, at which time the buffer was aspirated and 1 ml 0.5 M formic acid was added to each well. The formic acid lyses the cells, liberating soluble contents, and attaching most of the protein to the plates. The formic acid was left on the plates overnight then removed and lyophilized. 1 ml 0.5 M NaOH was then added to each well to solubilize the protein for determination of protein content in each well (BCA Protein assay kit, Pierce Chemical Co., Rockville IL). The lyophilized residues from each well were suspended in 0.3 ml of 100 mM sodium acetate buffer. pH 4.0, and assayed for CAMP by the protein kinase binding method of Gilman (19). Briefly, the reconstituted cell lysate, containing CAMP, was incubated with protein kinase (5 pg) and [3H]cAMP (2.9 nM) at pH 4.0, in a total volume of 0.3 ml. The incubation continued for 1 h on ice prior to filtration over glass fiber filters using a cell harvester (Brandel). Data was expressed as pmol per mg protein. To determine the rate of desensitization induced by the MSH analogs, the compound was added to the culture medium and left at 37°C for the desired time, ranging from 15 min to 24 h. After the appropriate length of time, the medium was aspirated, the cells were washed three times with 1 ml buffer, incubation buffer was added, and the normal CAMP experiment, as described above, was conducted. Results Non-transfected COS-7 cells, a simian kidney cell line, contain the mRNA encoding MC2R and MC3R, with receptor activation leading to an increase in CAMP accumulation in the intact cells
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MSH Receptor Desensitization
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(Bitler et al., submitted). Dose response curves with selected melanotropins or analogs indicated that the primary receptor in COS-7 cells functions differently than reported for most MCR activities. Although we can measure a very strong signal (up to a ten fold stimulation of CAMP levels) with aMSH and ACTH, the “superagonist” NDP-a-MSH clearly acts as a partial agonist in COS-7 cells (Fig. 1). The natural peptide ?I-MSH is significantly weaker, and did not reach maximal activity at the highest concentration tested, though it might at higher concentrations. This is not the case for NDP-a-MSH which clearly reaches a plateau by 1.O pM. However, as seen in table 1, even though it is a partial agonist in COS-7 cells, NDP-a-MSH maintains high affinity, producing half-maximal activity at approximately 7 nM, as compared to 22, 28 and 32 nM for the full agonists a-MSH, BMSH, and ACTH respectively.
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Pw#l M Fig 1 Stimulation of CAMP accumulation in COS-7 cells. Stimulation of CAMP accumulation was induced by a-MSH, (0); P-MSH, (0); ACTH, (v); NDP-aMSH, (V); and y-MSH (0). Because of variability in maximal CAMP accumulation on different days, results are presented as percent of maximal stimulation induced by a-MSH. Errors shown represent standard deviation from three separate experiments each conducted in duplicate.
In frog and lizard skin, the darkening induced by a-MSH reaches its maximal effect within 30-60 min in vitro, and 6 h in vivo, then returns to basal level within 2 h in vitro and 24 h in vivo. NDP-a-MSH-induced darkening of frog and lizard skin is still well above background after 8 h in vitro and 3 days in vivo (8.9). In COS-7 cells, the increase in CAMP induced by a-MSH is very rapid, reaching maximal stimulation within 5 min. Likewise, the duration of action is very short, returning to half of its maximal levels within 30 min (fig. 2a). In accordance with figure 1, a maximal concentration of NDP-a-MSH does not stimulate to the same extent as a-MSH. The duration of action, is also somewhat different. The time in which NDP-a-MSH reaches it maximal activity is slightly longer than that for a-MSH. Furthermore, if one plots CAMP as percent of maximal stimulation of each individual agonist, NDP-a-MSH retains its activity slightly longer than a-MSH (fig. 2b). However, because a-MSH stimulates to a greater extent, the actual amount of CAMP in the cells is still greater in the cells treated with CX-MSH. For most receptors, prolonged activity leads to desensitization of the response. If COS-7 cells are preincubated with one of the melanotropins, then washed to remove the peptide, the ability to produce a further stimulation of CAMP accumulation can be measured. As seen in figure 3a, Subsequent to a 5 min preincubation with NDP-a-MSH produces a very rapid desensitization. NDP-a-MSH, none of the melanotropins were able to produce significant stimulation of CAMP accumulation. Preincubation
with a-MSH
produces a similar, though not identical
effect.
a-MSH
also
MSH Receptor Desensitization
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produces desensitization, however, not as rapidly as NDP-a-MSH (fig. 3b). Although stimulation induced by subsequent addition of NDP-a-MSH and y-MSH is rapidly abolished, there is still TABLE 1 Potency of MSH Analogs for Stimulation of CAMP Accumulation
a-MSH
(4)
!ZZ:(::) NDP-a-MSH y-MSH (2)
in COS-7 Cells
21.8 I!Z8.5 27.8 32.5 I!I f 8.2 3.1 6.7 f 59 128 f25a
(3)
The measurement of the stimulation of CAMP accumulation was conducted as described in Methods. The number in parentheses indicate the number of individual experiments each conducted in duplicate. a. NDP-a-MSH and y-MSH did not stimulate CAMP accumulation to the same extent as a-MSH. EC50 values represent concentrations at which they reached half of their respective maximal activities. Average maximal CAMP accumulation were 47% for NDP-a-MSH and 5 1% for yMSH. 100 1ooE G E b !! P -
i ._ $ I 40-
$ 0
5 E 8 5 n
NDP-a-MSH 0,
, ( , , , 0 102030405060
I
NDP-a-MSH
80 60 40 20
0 0 102030405060
Minutes
Minutes
A
6 Fig 2
Time course of CAMP accumulation induced by a-MSH (0) and NDP-CXMSH (0). Values shown are (A) absolute amounts of CAMP in terms of pmol/mg cellular protein, and (B) CAMP in terms of percent maximal stimulation with respect to each MSH analog. The data shown was derived from three experiments each conducted in duplicate. and represents average values + standard deviation. considerable stimulation of CAMP accumulation pretreatment with a-MSH. After a 4 h pretreatment stimulate CAMP accumulation (data not shown).
induced by a-MSH and ACTH after 1 h with a-MSH, none of the analogs were able to
Discussion NDP-cr-MSH is a well-studied compound that has been shown to be an ultra-long lasting superagonist in a variety of species, whether measuring melanotropic activity or its rate limiting step,
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MSH Receptor Desensitization
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stimulation of tyrosinase activity, (8,9). There has been a great deal of speculation regarding the molecular mechanism of the prolonged activity. This speculation has ranged from this compound being stable in solution, to prolonged interaction with the receptor, to a direct effect on the signal transduction pathway (7). _ 1201 100 80 60 40 20 0
0
0 102030405060
102030405060 Minutes
Minutes B
A Fig 3
Rate of desensitization of CAMP accumulation induced by pretreatment with MSH analogs. CAMP accumulation was stimulated for 5 min, by a-MSH (1 PM), (0); NDP-a-MSH (0.2 jtM), (0); y-MSH (1 PM), (V); and ACTH (1 PM), (V); after pretreatment with (A) 1 pM NDP-a-MSH and (B) 1 pM a-MSH for the times indicated. The data shown was derived from three experiments each conducted in triplicate, and represents average values f standard deviation.
The MSH receptor signal is transduced through G,, inducing a stimulation of CAMP accumulation in cells containing the receptor, whether the receptor is expressed endogenously, or is transfected into the cells. The advantage of transfected cells is that the cells can contain a single, known, receptor type. In non-transfected cells that respond to MSH, such as COS-7 cells, not only is the receptor type present unknown, even the number of distinct types may be difficult to characterize. We have demonstrated by PCRLSouthern analysis that COS-7 cells have both MC2R and MC3R. and perhaps other known or as yet unidentified melanocortin receptors (Bitler et al.. submitted). The receptor in COS-7 cells that mediates the primary CAMP stimulatory activity is not clear. In each of the cloned receptors tested after transfection into a mammalian cell line, NDP-a-MSH has been shown to possess full agonist activity with respect to the naturally occurring MSH peptides (I 3,15,18). The finding that NDP-a-MSH shows partial agonist activity in COS-7 cells, but not in cells transfected with any of the known melanocortin receptors may be a function of the receptor number expressed in the transfected cells as compared to the number of receptors occurring naturally in COS-7 cells. It has been demonstrated that relative receptor activity can be a function of receptor number, and with a very high receptor reserve often found in transfected cells, a partial agonist can behave like a full agonist (20). The other possibility is that another, heretofore unidentified, melanotropin receptor is the primary mediator of activity in non-transfected COS-7 cells. This receptor could be derived from a new undiscovered gene, or possibly represent a posttranslational modification of one of the known MCRs. Nevertheless, the surprising finding that NDP-cx-MSH is a partial agonist, indicates that the receptor at which it is working has not been well characterized in the customary assays for melanotropin-like activity. One important property of partial agonists is the ability to produce antagonist actions. It is thereby possible that some of the biological activities attributed to this compound may be due to antagonist actions in viva. One advantage
of using a cell culture system for the study of receptor activation
is that the
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MSH Receptor Desensitization
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time course of activation, and the rate of desensitization can be easily determined. Based upon previous investigations into the nature of NDP-a-MSH, and other long lasting agonists, we expected to find prolonged activity in comparison to CK-MSH, with respect to the stimulation of CAMP accumulation. However, although the absolute amount of CAMP generated was different, the time course of the stimulation and disappearance of CAMP was quite similar for the two compounds (fig. 2). For each of the MSH analogs the time course of CAMP synthesis was very rapid, being complete within 5 min. The time course of CAMP stimulation by MSH in COS-7 cells is more rapid than that found by Adan et al. for MC3 receptors transfected into HEK 293 cells (20). Interestingly, even in the presence of the phosphodiesterase inhibitor IBMX, CAMP appears to be degraded very rapidly, or perhaps excreted from the cells, returning almost to basal levels within 1 h. In accordance with the rapid accumulation and reduction in CAMP levels subsequent to stimulation with a-MSH and NDP-a-MSH, preincubation with either compound leads to a rapid desensitization. However, a-MSH and NDP-a-MSH induce desensitization at different rates. A 510 min preincubation with NDP-a-MSH is sufficient to completely inhibit the stimulation of CAMP accumulation after subsequent stimulation with either a-MSH or NDP-a-MSH. Surprisingly, the full agonist a-MSH seems to produce desensitization at a slower rate. Even after I h preincubation with a-MSH, subsequent washing and reintroduction of a-MSH or ACTH produces some further stimulation of CAMP accumulation. One reason for the relatively rapid desensitization induced by NDP-a-MSH might be that it has considerably higher affinity for the receptor than a-MSH (see table 1) even though it is a partial agonist. Not only does NDP-a-MSH induce receptor desensitization more rapidly than a-MSH, it also desensitizes much more rapidly to a-MSH preincubation. This would be consistent with it being a partial agonist. In contrast to a full agonist, a partial agonist has no receptor reserve. If desensitization leads to the down regulation (i.e. a decrease) in receptor number (21), a partial agonist would be expected to have a decrease in activity prior to a full agonist. The rate of desensitization of this MCR is consistent with that of other G protein coupled rece tars. The process of desensitization has been extensively studied on a variety of receptors with the Ip-adrenergic receptor being the best characterized (22). It seems to be consistent with G-protein coupled receptors that desensitization is caused by phosphorylation either by a specific receptor kinase, such as P-adrenergic receptor kinase (PARK) (23), or less selective protein kinases such as protein kinase A (24), or protein kinase C (25). With the known MCRs, there are very few potential phosphorylation sites in the 3rd intracellular loop, or the C-terminal tail (16), common phosphorylation sites (22). Studies are now underway to identify the MCR that mediates the activity of the melanotropins in COS-7 cells, and to determine phosphorylation sites leading to receptor desensitization. References 1.
2. 3. 4. 5.
6. 7. 8. 9. 10. 11
J. CHALLIS and J.D. TOROSIS, Nature ?-> 269 8 18-820 (I 977). D. DE WIED and J. JOLLES, a,977-1059 (1982). Z. GALINA, Z. AMIT and J.M. VAN REE, Peptides, 6,285-291 (1985). S. LIN, C, CHAVES, E. WEIDEMANN, and M.H. HUMPHREY& M.H, Hypertension, lo, 619-627 (1987). C. DEBOLD, J.K. MENEFEE, W.E. NICHOLOSON, and D.N. ORTH, Mol. Endocr., 2, 862-870 (1988). C.H. Li., Recent Progress Hormone Res., l8, l-32 (1962). V.J. HRUBY, B.C. WILKES, W.L. CODY, T.K. SAWYER, and M.E. HADLEY, Peptide Protein Rev. 3, l-64 (1984). T.K. SAWYER, P.J. SANFILIPPO, V.J. HRUBY, M.H. ENGEL, C.B. HEWARD, J.B. BURNETT and M.E. HADLEY, Proc, Natl. Acad. Sci., 77,5754-5758 (1980). M.E. HADLEY, B. ANDERSON, C.B. HEWARD, T.K. SAWYER, and V.J. HRUBY, Science, 213, 1025-1027 (1981). M.E. HADLEY, Z.A. ABDEL MALEK, M.M. MARWAN, K.L. KREUTZFELD, and V.J. HRUBY, Endocrine Res, fl, 157-170 (1985). P.W. KREINER, C.J. GOLD, J.J. KEIRNS, W.A. BROCK, and Y. BITENSKY, J. Biol. Med., 465, 583-591 (1973).
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12. 13 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
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M.D. BREGMAN, T.K. SAWYER, M.E. HADLEY, and V.J. HRUBY, Arch. Biochem. Biophys., 201, l-7 (1980). K. MOUNTJOY, L.S. ROBBINS, M.T. MORTRUD, and R.D. CONE, Science, 257, 12481250 (1992). V. CHHAJLANI and J.E.S. WIKBERG, FEBS Lett., X&417-420 (1992). I. GANTZ, Y. KONDA, T. TASHIRO, Y. SHIMOTO, H. MIWA, G. MUNZERT, S.J. WATSON, J. DEL VALLE and T. YAMADA, J. Biol. Chem., &&8246-8250 (1993). I. GANTZ, H. MIWA, Y. KONDA, Y. SHIMOTO, T. TASHIRO, S.J. WATSON, J. DELVALLE andT. YAMADA, J. Biol. Chem., 268, 15174-15179 (1993b). V. CHHAIJLANI. V. R. MUCENIECE and J.E.S. WIKBERG, Biochem Biopohys Res. Commun., 195, 866-873 (1993). N. GRIFFON, V. MIGNON, P. FACCHINETTI, J. DIAZ, J.-C. SCHWARTZ AND P. SOKOLOFF, Biochem. Biophys. Res. Commun. 200, 1007-1014 (1994) A.G. GILMAN, Proc. Natl. Acad. Sci. USA, 67, 305-312 (1970). R.A.H. ADAN, R.D. CONE. J.P.H. BURBACH and W.H. GISPEN, Mol. Pharmacol. 46, 1182-l 190 (1994). D.J. MacEWAN, G.D. DIM, and G. MILLIGAN, Mol. Pharmacol. 48, 316-325 (1995). W.P. HAUSDORFF, M.G. CARON, and R.J. LEFKOWITZ, FASEB J., 4, 288 l-2889, (1990). J.L. BENOVIC, H. KUHN, I. WEYAND, J. CODINA, M.G. CARON, and R.J. LEFKOWITZ, Proc. Natl. Acad. Sci. USA, 84, 8879-8882 (1987). N.S. ROTH, P.T. CAMPBELL, M.G. CARON, R.J. LEFKOWITZ, and M.J. LOHSE, Proc. Natl. Acad. Sci. USA, 88, 620 l-6204 (199 1). M. BOUVIER, L.M.F. LEEB-LUNDBERG. J.L. BENOVIC, M.G. CARON, and R.J. LEFKOWITZ, J. Biol. Chem .1 -1 262 3106-3 113 (1987).
ELSEVIER
DESENSITIZATION
OF MELANOTROPIN
Life Science, Vol. 58, No. 15, pp. 1223-1229,19% Copyright0 1996 Elcevier Science Inc. Printed in the USA All rights ~cwed am-320.5/% sl5.00 + .oo
RECEPTORS
IN COS-7 CELLS’
Willma E. Polgar*, Catherine M. Bitler#, and Lawrence Toll* *Department of Neuroscience SRI International 333 Ravenswood Avenue Menlo Park CA 94025 #Neurex Corporation 3760 Haven Avenue, Menlo Park, CA 94025
(Received
in final form February 5, 19%)
Summary Non-transfected COS-7 cells have been found to possess functional melanotropin receptors on their cell surface. These receptors, and the properties of the melanocyte stimulating hormone (MSH) peptides can be characterized by measuring melanotropin stimulation of CAMP accumulation in the cells. In these cells we studied the ultra-long lasting super agonist [Nle4-D-Phe7]-cx-MSH (NDP-aMSH), and compared it with the endogenous MSH peptides with respect to potency, maximal activity, duration of action, and rate of desensitization. Surprisingly, NDPa-MSH did not act as a full agonist in COS-7 cells. In multiple experiments, it could stimulate CAMP accumulation to approximately 50% of the level of a-MSH, P-MSH and adrenocorticotropic hormone (ACTH). The MSH receptor mediating this activity is unknown. The time course of CAMP accumulation, and the duration of receptor activation was also investigated. In contrast to other systems, NDP-a-MSH did not induce prolonged activity, with respect to CAMP accumulation, in COS-7 cells. The MSH receptors present in COS-7 were found to desensitize rapidly subsequent to pretreatment by any of the MSH peptides. As expected for a partial agonist, the activity of NDP-a-MSH desensitized more rapidly than any of the full agonists. Surprisingly, desensitization induced by pretreatment with NDP-a-MSH also occurred more rapidly than desensitization induced by the other MSH analogs. Key Words: melanocyte stimulating hormone, melanotropin receptors, COW cells a-Melanotropin (cc-melanocyte stimulating hormone, a-MSH) and the other proopiomelanocortin (POMC)-derived peptides have been shown to have a variety of functions, ranging from well known effects on melanocytes and adrenal cortical cells, to more poorly understood central nervous system actions on consciousness, perception of pain, motivation and behavior (I -3). MSH has also been shown to effect natriuresis (4). POMC, or the mRNA encoding it, have been found in a variety of tissues including brain, lung, liver, adrenal, kidney, among others (5). A significant number of structure activity studies have been carried out on MSH and analogs, identifying structural requirements for binding to MSH and adrenocorticotropic hormone (ACTH) receptors (6.7). One of the most significant discoveries of the structure activity studies was the identification of a class of MSH analogs that display extremely high potency, and remarkably prolonged activity. The prototypical long-acting MSH analog is [Nle4-D-Phe7]-a-MSH (NDP-aMSH). This compound has been shown to have prolonged melanotropic activity in lizard and frog Corresponding Author: Lawrence Toll, SRI International, 333 Ravenswood Avenue, Menlo Park, CA 94025. USA. Telephone, (4 15) 859-380 1; FAX, (4 15) 859-4159; e-mail, toll @unix.sri.com. This work was supported by NIDA grant DA06682 to LT.
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skin in vitro and in viva (8,9). It also produces prolonged stimulation of tyrosinase activity in S91 melanoma cells. Interestingly, this compound induces tyrosinase activity after a very short exposure to the melanoma cells, and its stimulating activity is maintained after removal of the compound from the cells (10). There are other ways to measure activity of MSH and analogs. Receptor stimulation has been shown to mediate an increase in CAMP levels in S91 cells (11,12). Recently, five distinct melanotropin receptors have been cloned and transfected into a variety of cell lines (13-18). As expected, the melanotropins stimulate CAMP accumulation in cells transfected with any of the five melanotropin receptors (MCRs). More recently, we have demonstrated the presence of MCRs in non-transfected COS-7 cells. MSH, ACTH and various analogs mediate an increase in CAMP levels in this cell line, and mRNA encoding MC2R and MC3R have been demonstrated by Northern analysis and polymerase chain reaction (PCR)/Southern analysis (Bitler et al., submitted) In this study we have investigated the time course of the stimulation of CAMP accumulation, and the rate at which MSH and analogs induce desensitization of the CAMP-stimulating response of the melanotropin receptors found in COS-7 cells. We have found that MSH and analogs induce rapid desensitization of MCRs in COS-7 cells, and at least with respect to CAMP accumulation, that NDP-a-MSH is not a superagonist with prolonged activity in these cells. Methods Cells Culture COS-7 cells were grown on 100 mm culture dishes in Dulbeco’s Modified Eagle’s Medium (DMEM) supplemented with 5% fetal bovine serum and 10% horse serum. CAMP Assav COS-7 cells were plated on 6-well plates and used at confluence. Wells were washed once with incubation buffer containing: 130 mM NaCl, 4.8 mM KCI, 1.2 mM KH2P04, 1.3 mM CaCl2, 1.2 mM MgS04, 10 mM glucose, 50 mM isobutylmethylxanthine (IBMX) and 25 mM HEPES, pH 7.4. To each well was added 1 ml buffer, and cells were pre-incubated for 10 min at room temperature. This buffer was aspirated, and replaced with 1 ml fresh buffer containing the appropriate MSH peptide. To determine the time course of activity, cells were then incubated in triplicate for times ranging from 1 to 60 min, at which time the buffer was aspirated and 1 ml 0.5 M formic acid was added to each well. The formic acid lyses the cells, liberating soluble contents, and attaching most of the protein to the plates. The formic acid was left on the plates overnight then removed and lyophilized. 1 ml 0.5 M NaOH was then added to each well to solubilize the protein for determination of protein content in each well (BCA Protein assay kit, Pierce Chemical Co., Rockville IL). The lyophilized residues from each well were suspended in 0.3 ml of 100 mM sodium acetate buffer. pH 4.0, and assayed for CAMP by the protein kinase binding method of Gilman (19). Briefly, the reconstituted cell lysate, containing CAMP, was incubated with protein kinase (5 pg) and [3H]cAMP (2.9 nM) at pH 4.0, in a total volume of 0.3 ml. The incubation continued for 1 h on ice prior to filtration over glass fiber filters using a cell harvester (Brandel). Data was expressed as pmol per mg protein. To determine the rate of desensitization induced by the MSH analogs, the compound was added to the culture medium and left at 37°C for the desired time, ranging from 15 min to 24 h. After the appropriate length of time, the medium was aspirated, the cells were washed three times with 1 ml buffer, incubation buffer was added, and the normal CAMP experiment, as described above, was conducted. Results Non-transfected COS-7 cells, a simian kidney cell line, contain the mRNA encoding MC2R and MC3R, with receptor activation leading to an increase in CAMP accumulation in the intact cells
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MSH Receptor Desensitization
122.5
(Bitler et al., submitted). Dose response curves with selected melanotropins or analogs indicated that the primary receptor in COS-7 cells functions differently than reported for most MCR activities. Although we can measure a very strong signal (up to a ten fold stimulation of CAMP levels) with aMSH and ACTH, the “superagonist” NDP-a-MSH clearly acts as a partial agonist in COS-7 cells (Fig. 1). The natural peptide ?I-MSH is significantly weaker, and did not reach maximal activity at the highest concentration tested, though it might at higher concentrations. This is not the case for NDP-a-MSH which clearly reaches a plateau by 1.O pM. However, as seen in table 1, even though it is a partial agonist in COS-7 cells, NDP-a-MSH maintains high affinity, producing half-maximal activity at approximately 7 nM, as compared to 22, 28 and 32 nM for the full agonists a-MSH, BMSH, and ACTH respectively.
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-6
Pw#l M Fig 1 Stimulation of CAMP accumulation in COS-7 cells. Stimulation of CAMP accumulation was induced by a-MSH, (0); P-MSH, (0); ACTH, (v); NDP-aMSH, (V); and y-MSH (0). Because of variability in maximal CAMP accumulation on different days, results are presented as percent of maximal stimulation induced by a-MSH. Errors shown represent standard deviation from three separate experiments each conducted in duplicate.
In frog and lizard skin, the darkening induced by a-MSH reaches its maximal effect within 30-60 min in vitro, and 6 h in vivo, then returns to basal level within 2 h in vitro and 24 h in vivo. NDP-a-MSH-induced darkening of frog and lizard skin is still well above background after 8 h in vitro and 3 days in vivo (8.9). In COS-7 cells, the increase in CAMP induced by a-MSH is very rapid, reaching maximal stimulation within 5 min. Likewise, the duration of action is very short, returning to half of its maximal levels within 30 min (fig. 2a). In accordance with figure 1, a maximal concentration of NDP-a-MSH does not stimulate to the same extent as a-MSH. The duration of action, is also somewhat different. The time in which NDP-a-MSH reaches it maximal activity is slightly longer than that for a-MSH. Furthermore, if one plots CAMP as percent of maximal stimulation of each individual agonist, NDP-a-MSH retains its activity slightly longer than a-MSH (fig. 2b). However, because a-MSH stimulates to a greater extent, the actual amount of CAMP in the cells is still greater in the cells treated with CX-MSH. For most receptors, prolonged activity leads to desensitization of the response. If COS-7 cells are preincubated with one of the melanotropins, then washed to remove the peptide, the ability to produce a further stimulation of CAMP accumulation can be measured. As seen in figure 3a, Subsequent to a 5 min preincubation with NDP-a-MSH produces a very rapid desensitization. NDP-a-MSH, none of the melanotropins were able to produce significant stimulation of CAMP accumulation. Preincubation
with a-MSH
produces a similar, though not identical
effect.
a-MSH
also
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produces desensitization, however, not as rapidly as NDP-a-MSH (fig. 3b). Although stimulation induced by subsequent addition of NDP-a-MSH and y-MSH is rapidly abolished, there is still TABLE 1 Potency of MSH Analogs for Stimulation of CAMP Accumulation
a-MSH
(4)
!ZZ:(::) NDP-a-MSH y-MSH (2)
in COS-7 Cells
21.8 I!Z8.5 27.8 32.5 I!I f 8.2 3.1 6.7 f 59 128 f25a
(3)
The measurement of the stimulation of CAMP accumulation was conducted as described in Methods. The number in parentheses indicate the number of individual experiments each conducted in duplicate. a. NDP-a-MSH and y-MSH did not stimulate CAMP accumulation to the same extent as a-MSH. EC50 values represent concentrations at which they reached half of their respective maximal activities. Average maximal CAMP accumulation were 47% for NDP-a-MSH and 5 1% for yMSH. 100 1ooE G E b !! P -
i ._ $ I 40-
$ 0
5 E 8 5 n
NDP-a-MSH 0,
, ( , , , 0 102030405060
I
NDP-a-MSH
80 60 40 20
0 0 102030405060
Minutes
Minutes
A
6 Fig 2
Time course of CAMP accumulation induced by a-MSH (0) and NDP-CXMSH (0). Values shown are (A) absolute amounts of CAMP in terms of pmol/mg cellular protein, and (B) CAMP in terms of percent maximal stimulation with respect to each MSH analog. The data shown was derived from three experiments each conducted in duplicate. and represents average values + standard deviation. considerable stimulation of CAMP accumulation pretreatment with a-MSH. After a 4 h pretreatment stimulate CAMP accumulation (data not shown).
induced by a-MSH and ACTH after 1 h with a-MSH, none of the analogs were able to
Discussion NDP-cr-MSH is a well-studied compound that has been shown to be an ultra-long lasting superagonist in a variety of species, whether measuring melanotropic activity or its rate limiting step,
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MSH Receptor Desensitization
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stimulation of tyrosinase activity, (8,9). There has been a great deal of speculation regarding the molecular mechanism of the prolonged activity. This speculation has ranged from this compound being stable in solution, to prolonged interaction with the receptor, to a direct effect on the signal transduction pathway (7). _ 1201 100 80 60 40 20 0
0
0 102030405060
102030405060 Minutes
Minutes B
A Fig 3
Rate of desensitization of CAMP accumulation induced by pretreatment with MSH analogs. CAMP accumulation was stimulated for 5 min, by a-MSH (1 PM), (0); NDP-a-MSH (0.2 jtM), (0); y-MSH (1 PM), (V); and ACTH (1 PM), (V); after pretreatment with (A) 1 pM NDP-a-MSH and (B) 1 pM a-MSH for the times indicated. The data shown was derived from three experiments each conducted in triplicate, and represents average values f standard deviation.
The MSH receptor signal is transduced through G,, inducing a stimulation of CAMP accumulation in cells containing the receptor, whether the receptor is expressed endogenously, or is transfected into the cells. The advantage of transfected cells is that the cells can contain a single, known, receptor type. In non-transfected cells that respond to MSH, such as COS-7 cells, not only is the receptor type present unknown, even the number of distinct types may be difficult to characterize. We have demonstrated by PCRLSouthern analysis that COS-7 cells have both MC2R and MC3R. and perhaps other known or as yet unidentified melanocortin receptors (Bitler et al.. submitted). The receptor in COS-7 cells that mediates the primary CAMP stimulatory activity is not clear. In each of the cloned receptors tested after transfection into a mammalian cell line, NDP-a-MSH has been shown to possess full agonist activity with respect to the naturally occurring MSH peptides (I 3,15,18). The finding that NDP-a-MSH shows partial agonist activity in COS-7 cells, but not in cells transfected with any of the known melanocortin receptors may be a function of the receptor number expressed in the transfected cells as compared to the number of receptors occurring naturally in COS-7 cells. It has been demonstrated that relative receptor activity can be a function of receptor number, and with a very high receptor reserve often found in transfected cells, a partial agonist can behave like a full agonist (20). The other possibility is that another, heretofore unidentified, melanotropin receptor is the primary mediator of activity in non-transfected COS-7 cells. This receptor could be derived from a new undiscovered gene, or possibly represent a posttranslational modification of one of the known MCRs. Nevertheless, the surprising finding that NDP-cx-MSH is a partial agonist, indicates that the receptor at which it is working has not been well characterized in the customary assays for melanotropin-like activity. One important property of partial agonists is the ability to produce antagonist actions. It is thereby possible that some of the biological activities attributed to this compound may be due to antagonist actions in viva. One advantage
of using a cell culture system for the study of receptor activation
is that the
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time course of activation, and the rate of desensitization can be easily determined. Based upon previous investigations into the nature of NDP-a-MSH, and other long lasting agonists, we expected to find prolonged activity in comparison to CK-MSH, with respect to the stimulation of CAMP accumulation. However, although the absolute amount of CAMP generated was different, the time course of the stimulation and disappearance of CAMP was quite similar for the two compounds (fig. 2). For each of the MSH analogs the time course of CAMP synthesis was very rapid, being complete within 5 min. The time course of CAMP stimulation by MSH in COS-7 cells is more rapid than that found by Adan et al. for MC3 receptors transfected into HEK 293 cells (20). Interestingly, even in the presence of the phosphodiesterase inhibitor IBMX, CAMP appears to be degraded very rapidly, or perhaps excreted from the cells, returning almost to basal levels within 1 h. In accordance with the rapid accumulation and reduction in CAMP levels subsequent to stimulation with a-MSH and NDP-a-MSH, preincubation with either compound leads to a rapid desensitization. However, a-MSH and NDP-a-MSH induce desensitization at different rates. A 510 min preincubation with NDP-a-MSH is sufficient to completely inhibit the stimulation of CAMP accumulation after subsequent stimulation with either a-MSH or NDP-a-MSH. Surprisingly, the full agonist a-MSH seems to produce desensitization at a slower rate. Even after I h preincubation with a-MSH, subsequent washing and reintroduction of a-MSH or ACTH produces some further stimulation of CAMP accumulation. One reason for the relatively rapid desensitization induced by NDP-a-MSH might be that it has considerably higher affinity for the receptor than a-MSH (see table 1) even though it is a partial agonist. Not only does NDP-a-MSH induce receptor desensitization more rapidly than a-MSH, it also desensitizes much more rapidly to a-MSH preincubation. This would be consistent with it being a partial agonist. In contrast to a full agonist, a partial agonist has no receptor reserve. If desensitization leads to the down regulation (i.e. a decrease) in receptor number (21), a partial agonist would be expected to have a decrease in activity prior to a full agonist. The rate of desensitization of this MCR is consistent with that of other G protein coupled rece tars. The process of desensitization has been extensively studied on a variety of receptors with the Ip-adrenergic receptor being the best characterized (22). It seems to be consistent with G-protein coupled receptors that desensitization is caused by phosphorylation either by a specific receptor kinase, such as P-adrenergic receptor kinase (PARK) (23), or less selective protein kinases such as protein kinase A (24), or protein kinase C (25). With the known MCRs, there are very few potential phosphorylation sites in the 3rd intracellular loop, or the C-terminal tail (16), common phosphorylation sites (22). Studies are now underway to identify the MCR that mediates the activity of the melanotropins in COS-7 cells, and to determine phosphorylation sites leading to receptor desensitization. References 1.
2. 3. 4. 5.
6. 7. 8. 9. 10. 11
J. CHALLIS and J.D. TOROSIS, Nature ?-> 269 8 18-820 (I 977). D. DE WIED and J. JOLLES, a,977-1059 (1982). Z. GALINA, Z. AMIT and J.M. VAN REE, Peptides, 6,285-291 (1985). S. LIN, C, CHAVES, E. WEIDEMANN, and M.H. HUMPHREY& M.H, Hypertension, lo, 619-627 (1987). C. DEBOLD, J.K. MENEFEE, W.E. NICHOLOSON, and D.N. ORTH, Mol. Endocr., 2, 862-870 (1988). C.H. Li., Recent Progress Hormone Res., l8, l-32 (1962). V.J. HRUBY, B.C. WILKES, W.L. CODY, T.K. SAWYER, and M.E. HADLEY, Peptide Protein Rev. 3, l-64 (1984). T.K. SAWYER, P.J. SANFILIPPO, V.J. HRUBY, M.H. ENGEL, C.B. HEWARD, J.B. BURNETT and M.E. HADLEY, Proc, Natl. Acad. Sci., 77,5754-5758 (1980). M.E. HADLEY, B. ANDERSON, C.B. HEWARD, T.K. SAWYER, and V.J. HRUBY, Science, 213, 1025-1027 (1981). M.E. HADLEY, Z.A. ABDEL MALEK, M.M. MARWAN, K.L. KREUTZFELD, and V.J. HRUBY, Endocrine Res, fl, 157-170 (1985). P.W. KREINER, C.J. GOLD, J.J. KEIRNS, W.A. BROCK, and Y. BITENSKY, J. Biol. Med., 465, 583-591 (1973).
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