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Synthesis of a hormonally active conjugate of α-MSH, ferritin, and fluorescein
ANALYTICAL
BIOCHEMISTRY
Synthesis
84, 37-48 (1978)
of a Hormonally Active Conjugate Ferritin, and Fluorescein
ALBERT DIPASQUALE,JANOS M. VARGA,GISELA JOSEPH MCGUIRE Department
of Dermatology.
Yale University School Connecticut 06510
of CPMSH,
MOELLMANN,
of Medicine,
AND
Near, Haven,
Received August 6, 1976; accepted August 12, 1977 A conjugate composed ofa-MSH, ferritin, and fluorescein has been synthesized. The conjugate is biologically active: It is one-tenth as active as free LY-MSH in darkening frog skin and one-third as active as free (Y-MSH in stimulating tyrosinase activity in cultivated Cloudman melanoma cells. Binding of the conjugate to cultivated Cloudman melanoma cells was specific; binding was inhibited by at least 80% with either 5 x 10m5M (Y-or P-MSH. An average affinity constant of I .3 x IO9 liters/moI with approximately 1 to 4 x IO4sites/cell was calculated. The half-life of the conjugate-receptor complex is 58 min. The binding of the conjugate to cultivated Cloudman melanoma cells can be visualized by fluorescence microscopy. A patchy, perinuclear distribution of the conjugate, similar to that of FITC-PMSH. was observed. Electron microscopic examination revealed the conjugate to be at the cell surface.
Melanocyte-stimulating hormone (MSH) interacts with receptors at the cell surface of cultivated Cloudman melanoma cells (NCTC 3960 CCL 53) and activates adenylate cyclase (l-3). In order to locate the MSH-receptor complexes, a probe was needed that had the following characteristics: (i) specificity of binding to MSH receptors; (ii) visibility by transmission electron microscopy; (iii) visibility by light microscopy in order to select receptor-bearing cells for electron microscopy (4); (iv) retention of biological activity of the hormone, e.g., stimulation of tyrosinase activity in cultivated Cloudman melanoma cells and darkening of frog skin. We report here a technique for synthesizing a conjugate which has the above characteristics. It is composed of a-MSH, ferritin, and fluorescein isothiocyanate. In the first step, ferritin and fluorescein isothiocyanate are joined. MSH is then attached to the conjugate by the formation of a peptide bond to ferritin. The resultant molecule is biologically active and its binding to cultivated Cloudman melanoma cells is inhibited by either cr- or P-MSH. This work has been reported in preliminary form (5).
37
0003-2697/78/0841-0037$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
38
DlPASQUALE
MATERIALS Synthesis of a Ferritin -FITC
ET AL.
AND METHODS
Conjugate
Horse spleen ferritin recrystallized six times (Miles Laboratories) was recrystallized twice again wtih cadmium sulfate (6). The crystalline ferritin was redissolved at a concentration of 10 mg/ml in 2% ammonium sulfate and was centrifuged at 40,OOOg for 20 min. The supernatant was dialyzed against 0.9% NaCl overnight to remove the cadmium and then was dialyzed against 0.1 M NaHCO,, pH 8.3 overnight. Alternatively, cadmium was removed and the buffer was exchanged by chromatography on Sephadex G-25. The addition of 5 drops of 1 N NaOH to 5 ml of a solutionn of 10e8 M ferritin failed to show any precipitate of Cd(OH),, suggesting that less than 2.6 pg/ml of cadmium was present (7). The ferritin-containing fractions, which appeared in the void volume, were pooled and concentrated with an Amicon filter (XM 50, 43 mm) (8). Ferritin solutions (4-7 mg/ml) were sterilized by passage through a 0.45~pm Millipore filter and were stored at 4°C in the dark. Three milligrams of fluorescein isothiocyanate (FITC) (Sigma Chemical Co.) in 0.4 ml of 0.1 M NaHC03, pH 8.3, were added to 0.5 ml of 6-mg/ml ferritin solution. The mixture was stirred for 10 min at 25°C until clear, covered, stirred in the dark at 4°C for 72 h, and then eluted from a Sephadex G-25 column (0.52 x 32 cm) with 0.1 M NaHCO, (pH 8.3,4”C). The ferritin-FITC appeared in the void volume and the unreacted FITC was retained. The ratio of ferritin to FITC was calculated to be 1:6. This was calculated in the following way: Ferritin was passed through a column as described above, except that FITC was absent. The concentration of ferritin in the peak fractions was determined by absorbancy at 440 nm. Known concentrations of FITC were added to the ferritin. The fluorescence of FITC in the presence of ferritin was compared with the fluorescence of the peak fraction of ferritin-FITC, and the amount of FITC present per ferritin molecule was estimated from a standard curve. Synthesis of the a-MSH-Ferritin
-FITC
Conjugate
Immediately after elution, the ferritin-FITC (1 mg in 0.7 ml) was mixed with 3 mg of 1-ethyl-3-(3-diethylaminopropyl) carbodiimide (EDC) (Polysciences, Inc., Warrington, Pa.) in 100 ~1 of distilled water at 25°C. After 1 min, 10 pg of lz51-labeled-rw-MSH in 100 ~1 of PBS (0.15 M NaCl, 0.01 M sodium phosphate, pH 7.4) was added to the tube. [The lz51-labeled-a-MSH had a specific activity of 2.43 x lo4 cpm/pg and had been iodinated according to the method described for P-MSH (4).] One minute after the addition of lz51-labeled-cr-MSH, 1 mg of unlabeled a-MSH in 100 ~1 of distilled water was added. Labeling of ferritin-FITC with lz51-labeled-a-MSH was maximized by adding the unlabeled a-MSH after lz51-labeled-a-MSH to avoid
BIOLOGICALLY
ACTIVE
MARKER
FOR MSH
39
dilution. The number of a-MSH molecules per ferritin molecule was determined from syntheses in which the *251-labeled-a-MSH and unlabeled (r-MSH were added simultaneously. The reaction mixture was kept in the dark at 25°C for 20 min and then was eluted from a column of Sephadex G-100 (0.5 x 32 cm) with PBS, pH 7.4, at 4°C. Fractions of 0.2 ml were collected before the emergence of the dark brown ferritin-containing band. Radioactivity was measured in each fraction on an AW 1450 gamma counter (Scientific Products, Edison, N.J.). All a-MSH-ferritin-FITC used in these studies was prepared with 1251-labeled-a-MSH as well as with unlabeled a-MSH. The use of 1251-labeled-a-MSH permitted localization of bound and free a-MSH during chromatography. A scheme of the synthesis of the a-MSH-ferritin-FITC conjugate is shown in Fig. 1. Fluorescence was measured with a Farrand Mark I spectrofluorometer (Farrand Optical Co., Valhalla, N.Y.), and ferritin concentration was determined by absorbancy at 440 nm (1.5 OD = 1.O mg/ml of ferritin). The elution profile is shown in Fig. 2. In the peak fraction there are on the average five molecules of a-MSH and six molecules of FITC bound to each ferritin molecule. The number of MSH molecules on each ferritin molecule was calculated by assuming that the iodinated MSH and unlabeled MSH react with ferritinFITC at equal rates. This is probable since ferritin-FITC reacts with (YMSH at the amino group of lysine (amino acid No. 11) (9). Since a-MSH is iodinated at its tyrosine residue, the iodination should not influence the reaction rate. The yield of ferritin in the peak fraction is roughly 1014ferritin molecules/O.2 ml of PBS. The simultaneous elution of a-MSH with ferritin-FITC from a Sephadex G-100 column is not sufficient evidence that the molecules are covaH2N-
FIG. 1. Scheme of synthesis of the cY-MSH-ferritin-FITC conjugate. Fluorescein isothiocyanate (FITC) reacts with ferritin to produce ferritin-FITC. Ferritin-FITC then reacts with carbodiimide (EDCD) producing a compound which reacts with the amino group of MSH to produce an MSH-ferritin complex. An alternate reaction is possible in which the carboxyl group of MSH reacts with carbodiimide and the final MSH-ferritin complex is established with an amino group of ferritin; however this reaction is less likely because of the blocking by FITC of amino groups on the ferritin molecule.
DrPASQUALE
ET AL.
FRACTION
FIG. 2. Elution profile of a-MSH-ferritin-FITC. a-MSH-ferritin-FITC emerges in the void volume (7.5 ml). Each fraction contains 0.2 ml. The absorbancy of a 1: 10 dilution of each fraction was measured at 440 nm. Fluorescence of a 1: 10 dilution of each fraction was measured at an excitation wavelength of 480 nm and an emission wavelength of 515 nm using entrance and exit slits of 10 nm. (A) 9 counts per minute; (0) microamps; (x) OD at 440 nm.
lently bound. If noncovalent interactions were responsible for the conjugation, then the a-MSH would dissociate from the ferritin-FITC under conditions where a new equilibrium could be established. Biological activity of a-MSH freed from the noncovalently bound complex might then be mistaken for that of the complex. Two observations suggest that the complex is bound covalently. cr-MSH-ferritin-FITC is less electronegative than ferritin-FITC as determined by electrophoresis on agarose gels (Immunotee II IEP agarose plates, Behring Diagnostics, Somerville, N.J.) in barbital buffer (pH 8.2, ionic strength 0.04). When the conjugate is chromatographed on a Sephadex G-100 column equilibrated with PBS containing 0.5 M NaCI, only 6% of the a-MSH is dissociated from the conjugate and is eluted after the conjugate. On a third pass through a Sephadex G- 100 column in the presence of 0.5 M NaCl greater than 99% of the (r-MSH is excluded with the ferritin-FITC. RESULTS Tests of Binding Specificity
All binding experiments were performed at 0°C to avoid internalization of the conjugate by the cells. Cloudman melanoma cells (NCTC 3960 CCL
BIOLOGICALLY
ACTIVE
MARKER
FOR
MSH
41
53) were seeded at 5 x lo5 cells 25cm2 Corning flask in F-10 medium (GIBCO) supplemented with 2% fetal calf serum, 10% horse serum (mycoplasma free, Microbiological Associates), and 50 pg/ml of gentamycin (Schering Corp., Nutley, N.J.). Binding was measured either 24 or 48 hr after seeding while the cells were in their logarithmic growth phase. The medium was removed and the flasks (2-5 x lo5 cells) were rinsed twice with 35 ml of PBS (o”C, pH 7.4). cw-MSH-ferritin-FITC from a peak fraction was diluted with PBS, pH 7.4, and 20 pl(2 x 10-l’ to 10e7 M) was added to each drained flask. Molarity is expressed on the basis of ferritin. The flasks were then wrapped in aluminum foil and were kept at 0°C for 15 min [saturation of binding was reached in 15 min (Fig. 3)]. They were then rinsed five times with 15 ml per wash of PBS, pH 7.4, at 0°C and were drained. The duration of the washing procedure was less than 30 sec. One milliliter of 0.1 N NaOH was added to the flasks for 1 hr to dissolve the cells, which were then kept at 0°C in the dark. The intensity of fluorescence was directly proportional to the amount of conjugate present. Light scattering by the dissolved cells, which was proportional to cell number, was subtracted from the fluorescence readings. No significant quenching of fluorescence was caused by cellular material or NaOH. The fluorescence of the conjugate was unchanged by binding to the cell. Further, the FITC
FIG. 3. Kinetics of binding of conjugate. Cloudman melanoma cells were seeded at 5 x IO5 ceils/25-cm* Falcon flask 24 hr prior to the start of the experiments. Measurements of binding of conjugate were performed at 0°C as described in the text using entrance and exit slits of 20 nm on the fluorimeter. All measurements have been corrected for background fluorescence. (A) Rate of binding of conjugate at a concentration of 1 x IO-” M. Ordinate: molecules of conjugate bound (X lO-‘O)/5 x IO5 cells; abscissa: time in minutes. (B) Rate of dissociation of conjugate and receptors. Ordinate: molecules of conjugate bound (x IO-i”)/5 x IO5 cells (semilogarithmic plot); abscissa: time in minutes. (C) Competitive inhibition of binding of 5 x 10m9 M conjugate (0) or 5 X 10e9 M ferritin-FITC (0) by increasing concentrations of P-MSH [k, = 1.0 x IO* litersimol, 1 x 10’ receptors/cell, saturation within 15 min (4)l. A total of 20 ~1 of PBS containing appropriate concentrations of conjugate, ferritin-FITC, and P-MSH were added to flasks for 20 min at 0°C. Ordinate: molecules of conjugate or ferritinFITC bound (X lo-95 x 10-5cells: abscissa: molarity ofp-MSH (x 10% a-MSH at 5 x 1O-5 M inhibited binding of the conjugate to the same degree as did 5 x 10m5 M @-MSH.
42
DrPASQIJALE
ET AL.
remained associated with the conjugate after binding. When the melanoma cells were exposed to the conjugate for 15 min, washed extensively, dissolved in 0.1 N NaOH, neutralized with HCl, and passed through a Sephadex G-100 column in the presence of 0.5 M NaCl, all detectable fluorescence was eluted with the ferritin in the high molecular weight fractions; none appeared as free FITC. Additions of a-MSH-ferritin-FITC from lo-l1 to 3 x lo+ M (based on ferritin) showed a saturation at the latter concentration. In two experiments an average affinity constant (K,) of 1.3 x IO9 liters/m01 with approximately l-4 x lo4 sites/cell was calculated (Fig. 4). Binding in the concentration range from lo-lo to 6 x lOm8 M was specific; either (Y-or P-MSH eliminated 80-90% of the binding (Fig. 3C). At concentrations of lo+ M or higher, increasing nonspecific binding was observed. The ferritin-FITC molecule alone showed some binding at 0°C that could not be eliminated by high concentrations of pure (Y- or /3-MSH (Fig. 3C). Analysis of the kinetics of binding (Fig. 4) reveals an association rate constant (k,) of 3.0 x lo5 M-’ set-’ (derived from Fig. 4A using the formula k, = {2.303/[? (a - b)]} log b (a -x)/a (b -x), where a = [conjugate], b = [receptor], and x = amount of a bound at time t). Saturation occurs at 10 to 15 min. From Fig. 4B, a dissociation rate constant (k-,) of 2.35 x 10V4 set-’ was calculated. The half-life of the conjugate-receptor complex is approximately 58 min. Since the washing is completed in about 30 set, less than 0.8% of the label is lost during the washing procedure. Primary cultures of human skin fibroblasts bound 25% as much of the a-MSH-ferritin-FITC conjugate as melanoma cells. This binding was thought to be nonspecific since it could not be inhibited by high concentrations of (r-MSH or B-MSH. .I 5
FIG. 4. Binding of conjugate as a function of concentration. Cloudman melanoma cells were seeded at 5 x IO5 cells/25cm* Falcon flask 24 hr prior to the start of the experiment. Measurements of binding of conjugate were performed at 0°C as described in the text using entrance and exit slits of 20 nm on the tluorimeter. All measurements have been corrected for background fluorescence. Inset shows Scatchard plot of binding data.
BIOLOGICALLY
Tests of Biological
ACTIVE
MARKER
FOR MSH
43
Activity
Two different tests for biological activity were used: the darkening of frog skin and the stimulation of tyrosinase activity of cultivated Cloudman melanoma cells. Darkening offrog skin. Frog skin darkens rapidly in response to a-MSH. The change in color is caused by the intracellular translocation of melanin granules in dermal and, to a smaller extent, epidermal melanocytes. In lightened skin, the melanocytes appear punctate because the granules are aggregated in the cell body; in darkened skin, the melanocytes appear dentritic because the cell processes are filled with dispersed granules (10,ll). Table 1 shows the effect of ar-MSH-ferritin-FITC on skin darkening as measured by the changes in light reflectance. The a-MSH-ferritin-FITC molecule produced a change in reflectance approximately the same as that produced by a-MSH, although 10 times as much was required. Stimulation of tyrosinase activity. Cloudman mouse melanoma cells TABLE FROG
SKIN
DARKENING
Treatment PBS, pH 7.4, 25°C Ringer’s solution Ferritin (3 x 1Om8M) Ferritin-FITC (3 x IO-* M) FITC (3 x 1O-5 M) FITC (3 X IO-’ M) o-MSH-ferritin-FITC 3 X 1o-8 M 5 X lo-’ M 5 X to-” M 5 X lo-” M 5 X lo-” M a-MSH 3 X 1&8 M 5 X t&9 M 5 X lo-” M 5 X lo-” M 5 x 10-l* M
CAUSED
1 BY
(u-MSH-FERRITIN-FITC” Darkening Decrease in reflectance (%) after I hr
27 24 24 18 7 30 29 32 24 17
a Dorsal thigh skins of Rana pipiens were mounted on rings. They were rinsed with Ringer’s solution until light and then were immersed in Ringer’s solution in a small beaker with the dermis up. The Ringer’s solution was then removed from the dermal side of the chamber, and 0.4 ml of the solution to be tested was added into the well. Reflectance was measured as previously described (IO) with a Photovolt Model 670 reflectance meter. The molarity of the conjugate is based on ferritin.
44
DIPASQUALE
ET AL.
(NCTC 3960 CCL 53) respond to melanocyte-stimulating hormone with increases in tyrosinase activity (2), doubling time (2,3), and the median cell volume (3,12). These effects are mimicked by exposure to 3 x lo-* M cY-MSH-ferritin-FITC for 24 hr (Table 2). Dose-response curves of stimulation of tyrosinase activity in Cloudman melanoma cells exposed to a-MSH and a-MSH-ferritin-FITC are shown in Fig. 5. Half-maximal stimulation of tyrosinase is obtained with 1O-g M a-MSH and by 3 x lop9 M conjugate. The molarity of the conjugate is based on ferritin, and the concentration of MSH in the conjugate is fivefold that of ferritin. When the conjugate used in this experiment was chromatographed on a 0.5 M NaCl Sephadex column, less than 1% of the MSH could be dissociated. Accordingly, the activation of tyrosinase by the conjugate could not be attributed to free MSH. Incubation of the conjugate with a suspension of IO6 Cloudman melanoma cells in regular medium for 5 hr at 37°C did not separate (Y-MSH from the conjugate. This was determined by passage of the conjugate (removed from the suspension by centrifugation) through a Sephadex G- 100 column equilibrated with PBS containing 0.5 M NaCl and comparison of the elution of 1251-labeled-cr-MSH with the rust-colored conjugate. Thus it is unlikely that the stimulation of tyrosinase activity by the conjugate resulted from free a-MSH removed from the conjugate by enzyme activity in the serum or at the cell surface.
TABLE EFFECT
2
OF (u-MSH-FERRITIN-FITC ON TYROSINASE ACTIVITY, AND MEDIAN CELL VOLUME” Doubling time (hr)
Treatment Control a-MSH-ferritin-FITC a-MSH (3 x IO-”
(3 X IO-” M)
M)
26.0 34.8 36.0
DOUBLING
TIME,
Median cell volume (w+)
Tyrosinase activity (cpm/106 cells)
1,045 1,210 1,230
4,220 13,600 16,000
u NCTC 3960 CCL 53 Cloudman melanoma cells were seeded at 150,000 cells/25cm2 flask 24 hr before the start of the experiment. At 0 time MSH and L-[3,5-3H]tyrosine (0.25 &i/ml; 60 Ci/mmol, New England Nuclear) were added to triplicate flasks in a total volume of I.5 ml. After 48 hr, the medium was decanted, and the amount of 3H,0 released into the medium, a measure of tyrosinase activity (17), was assayed. An aliquot of 0.5 ml was mixed with 1.0 ml of 10% activated charcoal for 10 min at room temperature. After IO min the mixture was centrifuged at top speed (500 rpm) on a clinical centrifuge. An 0.5-ml aliquot of the clear supematant was passed through a 2.5-cm column of Dowex AG 5OW-X8 (200-400 mesh), and the column was rinsed once with 0.5 ml of distilled water. The total 1 ml eluted from the column was added to 10 ml of Aquasol and was counted in a Packard Tri-Carb liquid scintillation counter. Background (400 cpm) has been subtracted from all values. The molarity of the conjugate is based on ferritin.
BlOLOGICALLY
07 = 2 “0\ E a 0
12.000
-
8,000
-
6.000
-
-II
ACTIVE
MARKER
-10
45
FOR MSH
-9
-8
LOG [M] FIG. 5. Dose-response curves of stimulation of tyrosinase activity in Cloudman melanoma cells exposed to CX-MSH (x), a-MSH-ferritin-FITC (0). ferritin-FITC (A). Experiment and measurements were performed as described in Table 2. Control (0). Molarity of conjugate is based on ferritin.
Stability
The ratio of a-MSH:ferritin:FITC was 5: 1:6 immediately after chromatography of the reaction mixture. The same ratio was found after 7 days in the dark at 4°C when the conjugate was chromatographed again on a Sephadex G- 100 column and corrections were made for radioactive decay. Visualization by Fluorescence Microscopy of a-MSH-Ferritin Bound by Cultivated Cloudman Melanoma Cells
-FITC
Cloudman melanoma cells growing in Corning plastic flasks were exposed to 20 ~1 of a solution of MSH-ferritin-FITC for 15 min at 4°C. The solution was lOWEM with regard to ferritin. After washing with PBS, the cells were fixed in the dark for 30 min at 4°C in half-strength Karnovsky’s fixative (13) containing 0.1 M sucrose. Cells on plastic squares cut from the flask with a hot razor blade were examined under a fluorescence microscope using epi-illumination. As observed previously with FITC-/3-MSH (14), IO-20% of the cells showed significant fluorescence, which was patchy in distribution and usually located near the nucleus (Fig. 6). A similar distribution of *251-labeled-p-MSH had been found by autoradiography (15).
46
DIPASQUALE
ET AL.
FIG. 6. Patchy binding of cY-MSH-ferritin-FITC demonstrated by epifluorescence. Arrowheads indicate approximate margin of cell. Bar represents 20 pm.
Ultrastructural
Visualization
of a-MSH-Ferritin-FITC
Cloudman melanoma cells were exposed to cr-MSH-ferritin-FITC as described above and were fixed with half-strength Karnovsky’s fixative containing 0.1 M sucrose. After three rinses with 0.1 M sodium cacodylate buffer, the cells were scraped off the plastic growth surface with a rubber policeman, centrifuged into pellets; and refixed in osmium tetroxide. The cellular pellets were embedded in Epon and were sectioned on an LKB Ultrotome III. The ultrathin sections were stained with uranyl acetate and lead citrate and were examined under a Hitachi HU 11B electron microscope. A patch of ferritin bound at the cell surface is shown in Fig. 7. Significantly, such patches were present on only a few cells, as far as could be determined by viewing thin sections of cellular pellets. Ferritin was not found on cells exposed to ferritin alone or to ferritin-FITC. No conjugate was found to have been internalized. When cells exposed to a-MSH-ferritin-FITC were fixed and embedded in situ as a monolayer, fluorescent patches were apparent for about 12 hr after the Epon had hardened. The position of a single cell showing patchy fluorescence could be scored on the Epon with a diamond marker permitting subsequent electron microscopic examination of this cell. DISCUSSION
The a-MSH-ferritin-FITC complex described in this report can be used to locate MSH receptors at the ultrastructural level. It binds specifically to MSH receptors on the cell surface and is biologically active. Comparisons
BIOLOGICALLY
ACTIVE
MARKER
FOR MSH
47
FIG. 7. Patchy binding of a-MSH-ferritin-FITC demonstrated by electron microscopy. Arrowheads indicate ferritin on the cell surface. Nucleus (N) is in lower part of micrograph. Bar represents 0.4 pm.
of the activities of the conjugate vs free (Y-MSH are to some extent uninformative. When tyrosinase activity in cultured cells is measured, the activity of the conjugate is similar to that of free a-MSH. However, when the darkening of frog skin is assayed, approximately 10 times more conjugate than free a-MSH is needed to produce the same effect. This may be due to the relatively greater size of the conjugate, which penetrates the dermis less well than free (r-MSH. The apparent binding constant in cultivated Cloudman melanoma cells is higher than that reported for 1251-labeled-/3MSH (4); this may reflect the multivalence of the cu-MSH-ferritin-FITC conjugate. An increase in the number of ligands that can bind to MSH receptors would result in an increase in the apparent affinity of the conjugate to the cell surface (16). The similarity of binding characteristics between a-MSH-ferritinFITC, FITC-@MSH (14), and 1251-labeled-p-MSH (4) reinforces the conclusion that the complex is binding specifically to MSH receptors. The binding of each of these three moieties to a given cell is patchy whether viewed by light or electron microscopy. Since only IO-20% of cells in an asynchronous culture bind MSH (4) the fluorescence of the marker permits identification of MSH binding cells for subsequent electron microscopy. p-MSH-FITC has been reported to bind preferentially to the cell surface overlying the Golgi region (14). In electron micrographs of sedimented cells, we found several instances where a large number of ferritin particles had bound to those regions of the cell surface adjacent to the nucleus, occasionally in proximity to the Golgi apparatus. The functional implications of this localization (14) are being explored further.
48
DIPASQUALE
ET AL
ACKNOWLEDGMENTS We thank Elizabeth Godawska for preparing the melanoma cells for electron microscopy and for helping with the frog skin assays. The work was supported by U.S. Public Health Service Grant AM13929 and Training Grant USPHS 5 T32 AM 07016, American Cancer Society Grant BC-3J, and NSF Grant No. 61-43482. J.M.V. is a recipient of a Career Development Award from USPHS.
REFERENCES 1. 2. 3. 4.
Bitensky, M., and Demoupoulos, H. (1970) J. Invest. Dermatol. 54, 83. Wong, G., and Pawelek, J. (1973) Nature New Biol. 241, 213-215. DiPasquale, A., and McGuire, J. (1976) Exp. Cell Res. 102, 264-268. Varga, J., DiPasquale, A., Pawelek, J., McGuire, J., and Lerner, A. (1974) Proc. Nai. Acad. Sci. 71, USA 1590-1593. 5. DiPasquale, A., Varga, J. M., Moellmann, G., and McGuire, J. (1976) J. Cell Biol. 70, 423. 6.
7. 8. 9. 10. 11. 12. 13. 14.
Wu, M., and Davidson, N. (1973) J. Mol. Biol. 78, I-21. Breese, S., and Hsu, K. (1971) in Methods in Virology, (Maramorosch, K., and Koprowski, H., eds.), Vol. 5, pp. 399-422, Academic Press, New York. Heitzmann, H., and Richards, F. (1974) Proc. Nat. Acad. Sci. USA 71, 3537-3541. Lande, S., and Lerner, A. (1967) Phnrmacol. Rev. 19, l-20. Shizume, K., Lerner, A., and Fitzpatrick, T. (1954) Endocrinology 54, 553-560. Wright, P. (1955) Physiol. Zooi. 28, 204-218. DiPasquale, A., and McGuire, J. (1973) J. Cell Biol. 59, 82a. Karnovsky, M. (1965) J. Cell Biol. 27, 137a. Varga, J., Moellmann, G., Fritsch, P., Godawska, E., and Lerner, A. (1976) Proc. Nat. Acad.
Sci.
USA
73, 559-562.
15. Varga, J., Saper. M., Lerner, A., and Fritsch, P. (1976) J. Supramol. 16. Crothers, D., and Metzger, H. (1972) Immunochemistry 9, 341-357. 17. Pomerantz, S. (1966)J. Biol. Chem. 241, 161-168.
Struct.
4,45-49.
BIOCHEMISTRY
Synthesis
84, 37-48 (1978)
of a Hormonally Active Conjugate Ferritin, and Fluorescein
ALBERT DIPASQUALE,JANOS M. VARGA,GISELA JOSEPH MCGUIRE Department
of Dermatology.
Yale University School Connecticut 06510
of CPMSH,
MOELLMANN,
of Medicine,
AND
Near, Haven,
Received August 6, 1976; accepted August 12, 1977 A conjugate composed ofa-MSH, ferritin, and fluorescein has been synthesized. The conjugate is biologically active: It is one-tenth as active as free LY-MSH in darkening frog skin and one-third as active as free (Y-MSH in stimulating tyrosinase activity in cultivated Cloudman melanoma cells. Binding of the conjugate to cultivated Cloudman melanoma cells was specific; binding was inhibited by at least 80% with either 5 x 10m5M (Y-or P-MSH. An average affinity constant of I .3 x IO9 liters/moI with approximately 1 to 4 x IO4sites/cell was calculated. The half-life of the conjugate-receptor complex is 58 min. The binding of the conjugate to cultivated Cloudman melanoma cells can be visualized by fluorescence microscopy. A patchy, perinuclear distribution of the conjugate, similar to that of FITC-PMSH. was observed. Electron microscopic examination revealed the conjugate to be at the cell surface.
Melanocyte-stimulating hormone (MSH) interacts with receptors at the cell surface of cultivated Cloudman melanoma cells (NCTC 3960 CCL 53) and activates adenylate cyclase (l-3). In order to locate the MSH-receptor complexes, a probe was needed that had the following characteristics: (i) specificity of binding to MSH receptors; (ii) visibility by transmission electron microscopy; (iii) visibility by light microscopy in order to select receptor-bearing cells for electron microscopy (4); (iv) retention of biological activity of the hormone, e.g., stimulation of tyrosinase activity in cultivated Cloudman melanoma cells and darkening of frog skin. We report here a technique for synthesizing a conjugate which has the above characteristics. It is composed of a-MSH, ferritin, and fluorescein isothiocyanate. In the first step, ferritin and fluorescein isothiocyanate are joined. MSH is then attached to the conjugate by the formation of a peptide bond to ferritin. The resultant molecule is biologically active and its binding to cultivated Cloudman melanoma cells is inhibited by either cr- or P-MSH. This work has been reported in preliminary form (5).
37
0003-2697/78/0841-0037$02.00/O Copyright All rights
0 1978 by Academic Press. Inc. of reproduction in any form reserved.
38
DlPASQUALE
MATERIALS Synthesis of a Ferritin -FITC
ET AL.
AND METHODS
Conjugate
Horse spleen ferritin recrystallized six times (Miles Laboratories) was recrystallized twice again wtih cadmium sulfate (6). The crystalline ferritin was redissolved at a concentration of 10 mg/ml in 2% ammonium sulfate and was centrifuged at 40,OOOg for 20 min. The supernatant was dialyzed against 0.9% NaCl overnight to remove the cadmium and then was dialyzed against 0.1 M NaHCO,, pH 8.3 overnight. Alternatively, cadmium was removed and the buffer was exchanged by chromatography on Sephadex G-25. The addition of 5 drops of 1 N NaOH to 5 ml of a solutionn of 10e8 M ferritin failed to show any precipitate of Cd(OH),, suggesting that less than 2.6 pg/ml of cadmium was present (7). The ferritin-containing fractions, which appeared in the void volume, were pooled and concentrated with an Amicon filter (XM 50, 43 mm) (8). Ferritin solutions (4-7 mg/ml) were sterilized by passage through a 0.45~pm Millipore filter and were stored at 4°C in the dark. Three milligrams of fluorescein isothiocyanate (FITC) (Sigma Chemical Co.) in 0.4 ml of 0.1 M NaHC03, pH 8.3, were added to 0.5 ml of 6-mg/ml ferritin solution. The mixture was stirred for 10 min at 25°C until clear, covered, stirred in the dark at 4°C for 72 h, and then eluted from a Sephadex G-25 column (0.52 x 32 cm) with 0.1 M NaHCO, (pH 8.3,4”C). The ferritin-FITC appeared in the void volume and the unreacted FITC was retained. The ratio of ferritin to FITC was calculated to be 1:6. This was calculated in the following way: Ferritin was passed through a column as described above, except that FITC was absent. The concentration of ferritin in the peak fractions was determined by absorbancy at 440 nm. Known concentrations of FITC were added to the ferritin. The fluorescence of FITC in the presence of ferritin was compared with the fluorescence of the peak fraction of ferritin-FITC, and the amount of FITC present per ferritin molecule was estimated from a standard curve. Synthesis of the a-MSH-Ferritin
-FITC
Conjugate
Immediately after elution, the ferritin-FITC (1 mg in 0.7 ml) was mixed with 3 mg of 1-ethyl-3-(3-diethylaminopropyl) carbodiimide (EDC) (Polysciences, Inc., Warrington, Pa.) in 100 ~1 of distilled water at 25°C. After 1 min, 10 pg of lz51-labeled-rw-MSH in 100 ~1 of PBS (0.15 M NaCl, 0.01 M sodium phosphate, pH 7.4) was added to the tube. [The lz51-labeled-a-MSH had a specific activity of 2.43 x lo4 cpm/pg and had been iodinated according to the method described for P-MSH (4).] One minute after the addition of lz51-labeled-cr-MSH, 1 mg of unlabeled a-MSH in 100 ~1 of distilled water was added. Labeling of ferritin-FITC with lz51-labeled-a-MSH was maximized by adding the unlabeled a-MSH after lz51-labeled-a-MSH to avoid
BIOLOGICALLY
ACTIVE
MARKER
FOR MSH
39
dilution. The number of a-MSH molecules per ferritin molecule was determined from syntheses in which the *251-labeled-a-MSH and unlabeled (r-MSH were added simultaneously. The reaction mixture was kept in the dark at 25°C for 20 min and then was eluted from a column of Sephadex G-100 (0.5 x 32 cm) with PBS, pH 7.4, at 4°C. Fractions of 0.2 ml were collected before the emergence of the dark brown ferritin-containing band. Radioactivity was measured in each fraction on an AW 1450 gamma counter (Scientific Products, Edison, N.J.). All a-MSH-ferritin-FITC used in these studies was prepared with 1251-labeled-a-MSH as well as with unlabeled a-MSH. The use of 1251-labeled-a-MSH permitted localization of bound and free a-MSH during chromatography. A scheme of the synthesis of the a-MSH-ferritin-FITC conjugate is shown in Fig. 1. Fluorescence was measured with a Farrand Mark I spectrofluorometer (Farrand Optical Co., Valhalla, N.Y.), and ferritin concentration was determined by absorbancy at 440 nm (1.5 OD = 1.O mg/ml of ferritin). The elution profile is shown in Fig. 2. In the peak fraction there are on the average five molecules of a-MSH and six molecules of FITC bound to each ferritin molecule. The number of MSH molecules on each ferritin molecule was calculated by assuming that the iodinated MSH and unlabeled MSH react with ferritinFITC at equal rates. This is probable since ferritin-FITC reacts with (YMSH at the amino group of lysine (amino acid No. 11) (9). Since a-MSH is iodinated at its tyrosine residue, the iodination should not influence the reaction rate. The yield of ferritin in the peak fraction is roughly 1014ferritin molecules/O.2 ml of PBS. The simultaneous elution of a-MSH with ferritin-FITC from a Sephadex G-100 column is not sufficient evidence that the molecules are covaH2N-
FIG. 1. Scheme of synthesis of the cY-MSH-ferritin-FITC conjugate. Fluorescein isothiocyanate (FITC) reacts with ferritin to produce ferritin-FITC. Ferritin-FITC then reacts with carbodiimide (EDCD) producing a compound which reacts with the amino group of MSH to produce an MSH-ferritin complex. An alternate reaction is possible in which the carboxyl group of MSH reacts with carbodiimide and the final MSH-ferritin complex is established with an amino group of ferritin; however this reaction is less likely because of the blocking by FITC of amino groups on the ferritin molecule.
DrPASQUALE
ET AL.
FRACTION
FIG. 2. Elution profile of a-MSH-ferritin-FITC. a-MSH-ferritin-FITC emerges in the void volume (7.5 ml). Each fraction contains 0.2 ml. The absorbancy of a 1: 10 dilution of each fraction was measured at 440 nm. Fluorescence of a 1: 10 dilution of each fraction was measured at an excitation wavelength of 480 nm and an emission wavelength of 515 nm using entrance and exit slits of 10 nm. (A) 9 counts per minute; (0) microamps; (x) OD at 440 nm.
lently bound. If noncovalent interactions were responsible for the conjugation, then the a-MSH would dissociate from the ferritin-FITC under conditions where a new equilibrium could be established. Biological activity of a-MSH freed from the noncovalently bound complex might then be mistaken for that of the complex. Two observations suggest that the complex is bound covalently. cr-MSH-ferritin-FITC is less electronegative than ferritin-FITC as determined by electrophoresis on agarose gels (Immunotee II IEP agarose plates, Behring Diagnostics, Somerville, N.J.) in barbital buffer (pH 8.2, ionic strength 0.04). When the conjugate is chromatographed on a Sephadex G-100 column equilibrated with PBS containing 0.5 M NaCI, only 6% of the a-MSH is dissociated from the conjugate and is eluted after the conjugate. On a third pass through a Sephadex G- 100 column in the presence of 0.5 M NaCl greater than 99% of the (r-MSH is excluded with the ferritin-FITC. RESULTS Tests of Binding Specificity
All binding experiments were performed at 0°C to avoid internalization of the conjugate by the cells. Cloudman melanoma cells (NCTC 3960 CCL
BIOLOGICALLY
ACTIVE
MARKER
FOR
MSH
41
53) were seeded at 5 x lo5 cells 25cm2 Corning flask in F-10 medium (GIBCO) supplemented with 2% fetal calf serum, 10% horse serum (mycoplasma free, Microbiological Associates), and 50 pg/ml of gentamycin (Schering Corp., Nutley, N.J.). Binding was measured either 24 or 48 hr after seeding while the cells were in their logarithmic growth phase. The medium was removed and the flasks (2-5 x lo5 cells) were rinsed twice with 35 ml of PBS (o”C, pH 7.4). cw-MSH-ferritin-FITC from a peak fraction was diluted with PBS, pH 7.4, and 20 pl(2 x 10-l’ to 10e7 M) was added to each drained flask. Molarity is expressed on the basis of ferritin. The flasks were then wrapped in aluminum foil and were kept at 0°C for 15 min [saturation of binding was reached in 15 min (Fig. 3)]. They were then rinsed five times with 15 ml per wash of PBS, pH 7.4, at 0°C and were drained. The duration of the washing procedure was less than 30 sec. One milliliter of 0.1 N NaOH was added to the flasks for 1 hr to dissolve the cells, which were then kept at 0°C in the dark. The intensity of fluorescence was directly proportional to the amount of conjugate present. Light scattering by the dissolved cells, which was proportional to cell number, was subtracted from the fluorescence readings. No significant quenching of fluorescence was caused by cellular material or NaOH. The fluorescence of the conjugate was unchanged by binding to the cell. Further, the FITC
FIG. 3. Kinetics of binding of conjugate. Cloudman melanoma cells were seeded at 5 x IO5 ceils/25-cm* Falcon flask 24 hr prior to the start of the experiments. Measurements of binding of conjugate were performed at 0°C as described in the text using entrance and exit slits of 20 nm on the fluorimeter. All measurements have been corrected for background fluorescence. (A) Rate of binding of conjugate at a concentration of 1 x IO-” M. Ordinate: molecules of conjugate bound (X lO-‘O)/5 x IO5 cells; abscissa: time in minutes. (B) Rate of dissociation of conjugate and receptors. Ordinate: molecules of conjugate bound (x IO-i”)/5 x IO5 cells (semilogarithmic plot); abscissa: time in minutes. (C) Competitive inhibition of binding of 5 x 10m9 M conjugate (0) or 5 X 10e9 M ferritin-FITC (0) by increasing concentrations of P-MSH [k, = 1.0 x IO* litersimol, 1 x 10’ receptors/cell, saturation within 15 min (4)l. A total of 20 ~1 of PBS containing appropriate concentrations of conjugate, ferritin-FITC, and P-MSH were added to flasks for 20 min at 0°C. Ordinate: molecules of conjugate or ferritinFITC bound (X lo-95 x 10-5cells: abscissa: molarity ofp-MSH (x 10% a-MSH at 5 x 1O-5 M inhibited binding of the conjugate to the same degree as did 5 x 10m5 M @-MSH.
42
DrPASQIJALE
ET AL.
remained associated with the conjugate after binding. When the melanoma cells were exposed to the conjugate for 15 min, washed extensively, dissolved in 0.1 N NaOH, neutralized with HCl, and passed through a Sephadex G-100 column in the presence of 0.5 M NaCl, all detectable fluorescence was eluted with the ferritin in the high molecular weight fractions; none appeared as free FITC. Additions of a-MSH-ferritin-FITC from lo-l1 to 3 x lo+ M (based on ferritin) showed a saturation at the latter concentration. In two experiments an average affinity constant (K,) of 1.3 x IO9 liters/m01 with approximately l-4 x lo4 sites/cell was calculated (Fig. 4). Binding in the concentration range from lo-lo to 6 x lOm8 M was specific; either (Y-or P-MSH eliminated 80-90% of the binding (Fig. 3C). At concentrations of lo+ M or higher, increasing nonspecific binding was observed. The ferritin-FITC molecule alone showed some binding at 0°C that could not be eliminated by high concentrations of pure (Y- or /3-MSH (Fig. 3C). Analysis of the kinetics of binding (Fig. 4) reveals an association rate constant (k,) of 3.0 x lo5 M-’ set-’ (derived from Fig. 4A using the formula k, = {2.303/[? (a - b)]} log b (a -x)/a (b -x), where a = [conjugate], b = [receptor], and x = amount of a bound at time t). Saturation occurs at 10 to 15 min. From Fig. 4B, a dissociation rate constant (k-,) of 2.35 x 10V4 set-’ was calculated. The half-life of the conjugate-receptor complex is approximately 58 min. Since the washing is completed in about 30 set, less than 0.8% of the label is lost during the washing procedure. Primary cultures of human skin fibroblasts bound 25% as much of the a-MSH-ferritin-FITC conjugate as melanoma cells. This binding was thought to be nonspecific since it could not be inhibited by high concentrations of (r-MSH or B-MSH. .I 5
FIG. 4. Binding of conjugate as a function of concentration. Cloudman melanoma cells were seeded at 5 x IO5 cells/25cm* Falcon flask 24 hr prior to the start of the experiment. Measurements of binding of conjugate were performed at 0°C as described in the text using entrance and exit slits of 20 nm on the tluorimeter. All measurements have been corrected for background fluorescence. Inset shows Scatchard plot of binding data.
BIOLOGICALLY
Tests of Biological
ACTIVE
MARKER
FOR MSH
43
Activity
Two different tests for biological activity were used: the darkening of frog skin and the stimulation of tyrosinase activity of cultivated Cloudman melanoma cells. Darkening offrog skin. Frog skin darkens rapidly in response to a-MSH. The change in color is caused by the intracellular translocation of melanin granules in dermal and, to a smaller extent, epidermal melanocytes. In lightened skin, the melanocytes appear punctate because the granules are aggregated in the cell body; in darkened skin, the melanocytes appear dentritic because the cell processes are filled with dispersed granules (10,ll). Table 1 shows the effect of ar-MSH-ferritin-FITC on skin darkening as measured by the changes in light reflectance. The a-MSH-ferritin-FITC molecule produced a change in reflectance approximately the same as that produced by a-MSH, although 10 times as much was required. Stimulation of tyrosinase activity. Cloudman mouse melanoma cells TABLE FROG
SKIN
DARKENING
Treatment PBS, pH 7.4, 25°C Ringer’s solution Ferritin (3 x 1Om8M) Ferritin-FITC (3 x IO-* M) FITC (3 x 1O-5 M) FITC (3 X IO-’ M) o-MSH-ferritin-FITC 3 X 1o-8 M 5 X lo-’ M 5 X to-” M 5 X lo-” M 5 X lo-” M a-MSH 3 X 1&8 M 5 X t&9 M 5 X lo-” M 5 X lo-” M 5 x 10-l* M
CAUSED
1 BY
(u-MSH-FERRITIN-FITC” Darkening Decrease in reflectance (%) after I hr
27 24 24 18 7 30 29 32 24 17
a Dorsal thigh skins of Rana pipiens were mounted on rings. They were rinsed with Ringer’s solution until light and then were immersed in Ringer’s solution in a small beaker with the dermis up. The Ringer’s solution was then removed from the dermal side of the chamber, and 0.4 ml of the solution to be tested was added into the well. Reflectance was measured as previously described (IO) with a Photovolt Model 670 reflectance meter. The molarity of the conjugate is based on ferritin.
44
DIPASQUALE
ET AL.
(NCTC 3960 CCL 53) respond to melanocyte-stimulating hormone with increases in tyrosinase activity (2), doubling time (2,3), and the median cell volume (3,12). These effects are mimicked by exposure to 3 x lo-* M cY-MSH-ferritin-FITC for 24 hr (Table 2). Dose-response curves of stimulation of tyrosinase activity in Cloudman melanoma cells exposed to a-MSH and a-MSH-ferritin-FITC are shown in Fig. 5. Half-maximal stimulation of tyrosinase is obtained with 1O-g M a-MSH and by 3 x lop9 M conjugate. The molarity of the conjugate is based on ferritin, and the concentration of MSH in the conjugate is fivefold that of ferritin. When the conjugate used in this experiment was chromatographed on a 0.5 M NaCl Sephadex column, less than 1% of the MSH could be dissociated. Accordingly, the activation of tyrosinase by the conjugate could not be attributed to free MSH. Incubation of the conjugate with a suspension of IO6 Cloudman melanoma cells in regular medium for 5 hr at 37°C did not separate (Y-MSH from the conjugate. This was determined by passage of the conjugate (removed from the suspension by centrifugation) through a Sephadex G- 100 column equilibrated with PBS containing 0.5 M NaCl and comparison of the elution of 1251-labeled-cr-MSH with the rust-colored conjugate. Thus it is unlikely that the stimulation of tyrosinase activity by the conjugate resulted from free a-MSH removed from the conjugate by enzyme activity in the serum or at the cell surface.
TABLE EFFECT
2
OF (u-MSH-FERRITIN-FITC ON TYROSINASE ACTIVITY, AND MEDIAN CELL VOLUME” Doubling time (hr)
Treatment Control a-MSH-ferritin-FITC a-MSH (3 x IO-”
(3 X IO-” M)
M)
26.0 34.8 36.0
DOUBLING
TIME,
Median cell volume (w+)
Tyrosinase activity (cpm/106 cells)
1,045 1,210 1,230
4,220 13,600 16,000
u NCTC 3960 CCL 53 Cloudman melanoma cells were seeded at 150,000 cells/25cm2 flask 24 hr before the start of the experiment. At 0 time MSH and L-[3,5-3H]tyrosine (0.25 &i/ml; 60 Ci/mmol, New England Nuclear) were added to triplicate flasks in a total volume of I.5 ml. After 48 hr, the medium was decanted, and the amount of 3H,0 released into the medium, a measure of tyrosinase activity (17), was assayed. An aliquot of 0.5 ml was mixed with 1.0 ml of 10% activated charcoal for 10 min at room temperature. After IO min the mixture was centrifuged at top speed (500 rpm) on a clinical centrifuge. An 0.5-ml aliquot of the clear supematant was passed through a 2.5-cm column of Dowex AG 5OW-X8 (200-400 mesh), and the column was rinsed once with 0.5 ml of distilled water. The total 1 ml eluted from the column was added to 10 ml of Aquasol and was counted in a Packard Tri-Carb liquid scintillation counter. Background (400 cpm) has been subtracted from all values. The molarity of the conjugate is based on ferritin.
BlOLOGICALLY
07 = 2 “0\ E a 0
12.000
-
8,000
-
6.000
-
-II
ACTIVE
MARKER
-10
45
FOR MSH
-9
-8
LOG [M] FIG. 5. Dose-response curves of stimulation of tyrosinase activity in Cloudman melanoma cells exposed to CX-MSH (x), a-MSH-ferritin-FITC (0). ferritin-FITC (A). Experiment and measurements were performed as described in Table 2. Control (0). Molarity of conjugate is based on ferritin.
Stability
The ratio of a-MSH:ferritin:FITC was 5: 1:6 immediately after chromatography of the reaction mixture. The same ratio was found after 7 days in the dark at 4°C when the conjugate was chromatographed again on a Sephadex G- 100 column and corrections were made for radioactive decay. Visualization by Fluorescence Microscopy of a-MSH-Ferritin Bound by Cultivated Cloudman Melanoma Cells
-FITC
Cloudman melanoma cells growing in Corning plastic flasks were exposed to 20 ~1 of a solution of MSH-ferritin-FITC for 15 min at 4°C. The solution was lOWEM with regard to ferritin. After washing with PBS, the cells were fixed in the dark for 30 min at 4°C in half-strength Karnovsky’s fixative (13) containing 0.1 M sucrose. Cells on plastic squares cut from the flask with a hot razor blade were examined under a fluorescence microscope using epi-illumination. As observed previously with FITC-/3-MSH (14), IO-20% of the cells showed significant fluorescence, which was patchy in distribution and usually located near the nucleus (Fig. 6). A similar distribution of *251-labeled-p-MSH had been found by autoradiography (15).
46
DIPASQUALE
ET AL.
FIG. 6. Patchy binding of cY-MSH-ferritin-FITC demonstrated by epifluorescence. Arrowheads indicate approximate margin of cell. Bar represents 20 pm.
Ultrastructural
Visualization
of a-MSH-Ferritin-FITC
Cloudman melanoma cells were exposed to cr-MSH-ferritin-FITC as described above and were fixed with half-strength Karnovsky’s fixative containing 0.1 M sucrose. After three rinses with 0.1 M sodium cacodylate buffer, the cells were scraped off the plastic growth surface with a rubber policeman, centrifuged into pellets; and refixed in osmium tetroxide. The cellular pellets were embedded in Epon and were sectioned on an LKB Ultrotome III. The ultrathin sections were stained with uranyl acetate and lead citrate and were examined under a Hitachi HU 11B electron microscope. A patch of ferritin bound at the cell surface is shown in Fig. 7. Significantly, such patches were present on only a few cells, as far as could be determined by viewing thin sections of cellular pellets. Ferritin was not found on cells exposed to ferritin alone or to ferritin-FITC. No conjugate was found to have been internalized. When cells exposed to a-MSH-ferritin-FITC were fixed and embedded in situ as a monolayer, fluorescent patches were apparent for about 12 hr after the Epon had hardened. The position of a single cell showing patchy fluorescence could be scored on the Epon with a diamond marker permitting subsequent electron microscopic examination of this cell. DISCUSSION
The a-MSH-ferritin-FITC complex described in this report can be used to locate MSH receptors at the ultrastructural level. It binds specifically to MSH receptors on the cell surface and is biologically active. Comparisons
BIOLOGICALLY
ACTIVE
MARKER
FOR MSH
47
FIG. 7. Patchy binding of a-MSH-ferritin-FITC demonstrated by electron microscopy. Arrowheads indicate ferritin on the cell surface. Nucleus (N) is in lower part of micrograph. Bar represents 0.4 pm.
of the activities of the conjugate vs free (Y-MSH are to some extent uninformative. When tyrosinase activity in cultured cells is measured, the activity of the conjugate is similar to that of free a-MSH. However, when the darkening of frog skin is assayed, approximately 10 times more conjugate than free a-MSH is needed to produce the same effect. This may be due to the relatively greater size of the conjugate, which penetrates the dermis less well than free (r-MSH. The apparent binding constant in cultivated Cloudman melanoma cells is higher than that reported for 1251-labeled-/3MSH (4); this may reflect the multivalence of the cu-MSH-ferritin-FITC conjugate. An increase in the number of ligands that can bind to MSH receptors would result in an increase in the apparent affinity of the conjugate to the cell surface (16). The similarity of binding characteristics between a-MSH-ferritinFITC, FITC-@MSH (14), and 1251-labeled-p-MSH (4) reinforces the conclusion that the complex is binding specifically to MSH receptors. The binding of each of these three moieties to a given cell is patchy whether viewed by light or electron microscopy. Since only IO-20% of cells in an asynchronous culture bind MSH (4) the fluorescence of the marker permits identification of MSH binding cells for subsequent electron microscopy. p-MSH-FITC has been reported to bind preferentially to the cell surface overlying the Golgi region (14). In electron micrographs of sedimented cells, we found several instances where a large number of ferritin particles had bound to those regions of the cell surface adjacent to the nucleus, occasionally in proximity to the Golgi apparatus. The functional implications of this localization (14) are being explored further.
48
DIPASQUALE
ET AL
ACKNOWLEDGMENTS We thank Elizabeth Godawska for preparing the melanoma cells for electron microscopy and for helping with the frog skin assays. The work was supported by U.S. Public Health Service Grant AM13929 and Training Grant USPHS 5 T32 AM 07016, American Cancer Society Grant BC-3J, and NSF Grant No. 61-43482. J.M.V. is a recipient of a Career Development Award from USPHS.
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Bitensky, M., and Demoupoulos, H. (1970) J. Invest. Dermatol. 54, 83. Wong, G., and Pawelek, J. (1973) Nature New Biol. 241, 213-215. DiPasquale, A., and McGuire, J. (1976) Exp. Cell Res. 102, 264-268. Varga, J., DiPasquale, A., Pawelek, J., McGuire, J., and Lerner, A. (1974) Proc. Nai. Acad. Sci. 71, USA 1590-1593. 5. DiPasquale, A., Varga, J. M., Moellmann, G., and McGuire, J. (1976) J. Cell Biol. 70, 423. 6.
7. 8. 9. 10. 11. 12. 13. 14.
Wu, M., and Davidson, N. (1973) J. Mol. Biol. 78, I-21. Breese, S., and Hsu, K. (1971) in Methods in Virology, (Maramorosch, K., and Koprowski, H., eds.), Vol. 5, pp. 399-422, Academic Press, New York. Heitzmann, H., and Richards, F. (1974) Proc. Nat. Acad. Sci. USA 71, 3537-3541. Lande, S., and Lerner, A. (1967) Phnrmacol. Rev. 19, l-20. Shizume, K., Lerner, A., and Fitzpatrick, T. (1954) Endocrinology 54, 553-560. Wright, P. (1955) Physiol. Zooi. 28, 204-218. DiPasquale, A., and McGuire, J. (1973) J. Cell Biol. 59, 82a. Karnovsky, M. (1965) J. Cell Biol. 27, 137a. Varga, J., Moellmann, G., Fritsch, P., Godawska, E., and Lerner, A. (1976) Proc. Nat. Acad.
Sci.
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73, 559-562.
15. Varga, J., Saper. M., Lerner, A., and Fritsch, P. (1976) J. Supramol. 16. Crothers, D., and Metzger, H. (1972) Immunochemistry 9, 341-357. 17. Pomerantz, S. (1966)J. Biol. Chem. 241, 161-168.
Struct.
4,45-49.