Proc. Nat. Acad. Sci. USAVol. 73, No. 2, pp. 559-562, February 1976Cell Biology

Association of cell surface receptors for melanotropin with the Golgiregion in mouse melanoma cells

(melanocyte stimulating hormone/Golgi complex/melanization/fluorescence microscopy/double labeling)

JANOS M. VARGA, GISELA MOELLMANN, PETER FRITSCH, ELIZABETH GODAWSKA,AND AARON B. LERNERDepartment of Dermatology, Yale University School of Medicine, New Haven, Connecticut 06510

Contributed by Aaron B. Lerner, December 1, 1975

ABSTRACT Binding of 4-melanotropin (f3-melanocytestimulating hormone) to mouse melanoma cells occurs in aregion on the cell surface overlying the Golgi complex. Thisassociation was demonstrated by labeling cells with fluo-rescein isothiocyanate hormone and by locating the Golgicomplex with a histochemical test for thiamine pyrophospha-tase activity. The biologically active fluorescent horimone ap-pears on the surface and later in vesicles in the melanizedcells, as judged by fluorescence microscopy. It is conceivablethat internalization of the hormone is instrumental in theprocess of hormonally induced melanization. Because initialand late events of hormonally induced pigmentation are re-lated to the Golgi complex, it is likely that instructions thatfollow the attachment of melanotropin to receptors are car-ried out in a compartmentalized manner.

f3-Melanotropin (fl-melanocyte stimulating hormone; MSH)induces an increase in intracellular adenosine 3':5'-cyclicmonophosphate (cAMP), tyrosinase activity, and melanincontent in a line of cells from a mouse melanoma (1). Themajor known steps of this stimulation are: (a) recognition ofMSH by a receptor on the cell surface (2); (b) stimulation ofadenylate cyclase that results in an immediate rise in the in-tracellular concentration of cAMP (3); and (c) after a fewhours of delay, activation of tyrosinase (4, 5). We have re-cently demonstrated by light microscopic autoradiographythat the labeled hormone binds to mouse melanoma cells indiscrete areas on the cell surface (6). Binding of 125I-labeledMSH usually occurred as a single spot on the cell surface inthe perinuclear area. The similarity of the pattern of MSHbinding and the location of the Golgi complex, as deter-mined by a cytochemical test (8, 10), prompted us to investi-gate the relationship between the Golgi region and receptorsfor MSH. In this paper we present results obtained by a dou-ble-labeling technique that shows that the binding of MSHtakes place on the surface of the cells at sites related to theGolgi complex. Furthermore, we were able to demonstratethat the labeled hormone appears internalized in vesicleswithin the melanized cells.

MATERIALS AND METHODSA pigmented subclone (PSI-wt) of cells from the CloudmanS-91 (clone M-3, CCL 53.1) was cultured as described pre-viously (7). Cultures of cells on cover slips were used 40 hrafter inoculation. The cells were labeled at 00 with 125I-la-beled MSH according to published procedures (2). We usedour bubble method for light microscopic autoradiography(6). The binding of 125-labeled MSH showed a characteristic

Abbreviations: MSH, fl-melanotropin (f3-melanocyte stimulatinghormone) (Asp-Glu-Gly-Pro-Tyr-Lys-Met-Glu-His-Phe-Arg-Trp-Gly-Ser-Pro-Lys-Asp); cAMP, adenosine 3':5'-cyclic monophos-phate; phosphate-buffered saline, 0.01 M Na phosphate-0.15 MNaCl, pH 7.4; TPPase, thiamine pyrophosphatase; dopa, 3,4-dihy-droxy-phenylalanine; FITC, fluorescein isothiocyanate.

patchy distribution. The Golgi complex was labeled bymeans of a histochemical test for thiamine pyrophosphataseactivity (8, 10).Preparation of Fluorescein Isothiocyanate (FITC-

MSH. f3-MSH (3.75 mg) (kindly supplied by Dr. Saul Landeof this department) was dissolved in 1 ml of phosphate-buff-ered saline (pH 9.5, NaOH). The solution was stirred for 4hr with 0.5 mg of FITC at 40, dialyzed overnight with phos-phate-buffered saline (0.01 M Na phosphate-0.15 M NaCl,pH 7.4), and passed through a 0.8 X 40 cm column of Se-phadex G-10 equilibrated with phosphate-buffered saline.The FITC-MSH conjugate appeared in the void volume.The labeled hormone retained at least 95% of its original bi-ological activity.Double Labeling for MSH Receptors and for Thiamine

Pyrophosphatase (TPPase) Activity. Cells were fixed for 90sec at 220 with a mixture of 1.25% glutaraldehyde and 1%paraformaldehyde (9). The thiamine pyrophosphatase reac-tion (8, 10) was carried out first, followed by an incubationfor 15 min with FITC-MSH at 37°. The cells were washedthree times with phosphate-buffered saline, briefly rinsed indistilled water, and dried in air.

Detection of Internalized FITC-MSH. FITC-MSH (10-7M) was added to coverslip cultures of melanoma cells. Thecells were cultivated for an additional 30 hr in the presenceof FITC-MSH. The cultures were washed with phosphate-buffered saline (00), fixed with paraformaldehyde (1%, 0°),and after Giemsa staining they were air dried. Cells wereobserved in the fluorescence microscope without coverslipwith a 100 X oil immersion objective.

Localization of TPPase Activity with the Electron Mi-croscope. Cells grown in monolayers were detached fromthe culture dishes with 0.1 M EDTA and centrifuged. Thecellular pellets were rinsed with 0.1 M cacodylate buffer pH7.2, containing 5% sucrose; they were fixed with 3% glutar-aldehyde in sodium cacodylate buffer pH 7.2 for 30 min at40. The cells were incubated in the cytochemical medium(8, 10) for 1 hr at room temperature. The pellets werewashed again in buffer, immersed in osmium tetroxide, de-hydrated in ethanol, and embedded in Epon. Ultrathin sec-tions were double stained with uranyl acetate and lead ci-trate. The sections were examined in a Hitachi HU liB elec-tron microscope.

RESULTSTPPase as a Marker for the Golgi Complex. Thiamine

pyrophosphatase activity has been previously localized bylight microscopy in the B-16 strain of melanoma cells (10).This enzyme activity is restricted to the Golgi complex, ascan be seen from electron microscopic enzyme cytochemis-try on cells of the Cloudman strain (Fig. 1). The light micro-

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560 Cell Biology: Varga et al.

FIG. 1. Electron microscopic localization of TPPase activity. Arrows indicate the location of reaction product in lamellae of the Golgiapparatus. Magnification: 9600X. Insert: Light microscopic visualization of TPPase activity. Magnification: 310X.

scopic distribution of the TPPase reaction product is demon-strated in the inserted photomicrograph. In agreement withfindings on B-16 melanoma cells (10), the TPPase activity inthe Cloudman strain is evident most frequently in a singlefocus or very few perinuclear spots.Double Labeling for MSH Receptors and Golgi Com-

plex. We have reported earlier that receptors for MSH aredisplayed in a nonrandom manner on the surface of mousemelanoma cells (6). Autoradiographic labeling of the recep-tors and locating the Golgi complex by the TPPase reactionyielded almost identical images. Therefore, we developed amethod for labeling the MSH receptors with FITC-MSH.This test enabled us to visualize the Golgi area and MSH re-ceptors on the same cell, at the same time. The distributionof binding of the FITC-MSH conjugate to the surface ofmelanoma cells was similar to that seen with 125I-labeledMSH (see ref. 6 and Fig. 2). Binding of FITG-MSH most fre-quently occurred in the same area where the TPPase reac-tion outlined the location of the Golgi complex (Fig. 3). Onthe average, 12% of the total cell population was labeledwith FITG-MSH. The low labeling index is expected, be-cause only cells in the G-2 phase have active receptors (2).The frequency of cells carrying detectable TPPase reactionwas 35%. The FITG-MSH or TPPase related spots occupied2-8% of the total cell surface (see Figs. 1-3). An average of32% of the FITC-MSH labeled cells showed coincidental la-beling with TPPase. This is significant, since on the basis oflabeling frequencies and the relative size of the spots onlyone cell in 400 would be expected to be doubly labeled if co-incidental labeling were a chance event.

Internalization of FITC-MSH. When FITC-MSH wasadded to growing cultures of melanoma cells, two types oflabeling patterns were observed. Nonmelanized cells werelabeled on the surface in patches with diffuse border lines,similarly to Fig. 2. (The contours of the labeled cells are visi-

bly out of focus when the microscope is focused on thelabel.) In about 20% of the melanized cells the label ap-peared in small patches with sharp border lines. Thesepatches appeared to be located inside the cells (Fig. 4). Notethat the nucleus and melanized area are in focus, as well asthe fluorescent spots.

DISCUSSIONSeveral explanations may be offered to account for the pres-ence of receptors for MSH overlying the Golgi complex. (a)The relationship could be a chance coincidence. The proba-bility of such a coincidence is, however, about two orders ofmagnitude lower than the observed frequency of the doublelabeling. (b) The Golgi complex could be a "universal sink"

FIG. 2. Localization ofMSH receptors by FITC-MSH and flu-orescence microscopy. Arrow points to the position of FITC-MSHlabel. Photomicrograph was taken by transmission illuminationwith visible light and simultaneous fluorescence epi-illumination.Illumination by visible light was decreased to the point where bothcell morphology and the fluorescence marker could be visualized.Magnification: 400X.

Proc. Nat. Acad. Sci. USA 73 (1976)

Proc. Nat. Acad. Sci. USA 73 (1976) 561

FIG. 3. Double labeling of MSH receptors by FITC-MSH andthe Golgi complex by the TPPase reaction. The bigger arrowpoints to the position of FITC-MSH label and the smaller one tothe TPPase reaction product. The photomicrograph was takenwith a Zeiss transmitted-light fluorescence photomicroscope,equipped with mercury burner. Magnification: 630X.

in melanoma cells, taking up all kinds of labeled substanceswithout discrimination. The fact that we have not obtainedGolgi-related label with all markers, e.g., FITC-anti-rabbitantibodies alone did not label the Golgi region (unpublishedobservation), is an argument against this possibility. (c) MSHreceptors, like other polypeptide hormone receptors, are as-sumed to be glycoproteins (11). As such, they may emergefrom the Golgi complex (12). Their appearance in an areathat includes the Golgi complex might indicate their site ofsynthesis only. In such a case, their confinement to the Golgiregion would be temporary. We have found, in contrast,that MSH receptors do not spread over the whole cell sur-face; they remain as patches (6). (d) MSH receptors may bepermanently associated with an area that is related to theGolgi apparatus. We favor this latter possibility and assumethat such an association would have a functional signifi-cance.Our finding, that the hormone binds in a region overlying

the Golgi complex, together with the known intracellular lo-calization of tyrosinase activity and melanin deposition (13,14), suggest that the execution of the hormonal message isconfined to the Golgi region.Our working hypothesis is depicted in Fig. 5. The process

of MSH-induced melanization may be subdivided into aMSH-independent preparatory phase and the actual execu-tion of hormonal instruction. During the preparatoryphase, premelanosomes are formed in the smooth endoplas-mic reticulum in the proximity of the Golgi region (13). Ty-rosinase is synthesized in the rough endoplasmic reticulum

FIG. 4. Internalization of FITC-MSH. Arrow points to ves-icles, containing FITC-MSH. Magnification: 1250X.

FIG. 5. A scheme for MSH-induced melanization. T = inactivetyrosinase; T* = active tyrosinase; RER = endoplasmic reticulum;SER = smooth endoplasmic reticulum; MS = melanosome; pMS =premelanosome; MG = melanin granule; U = "empty" MSH re-ceptor-adenylate cyclase; 0 = MSH receptor-MSH-activated ade-nylate cyclase complex.

(14) and reaches the Golgi complex (10), where a carbohy-drate moiety may be attached to it (15). These moleculesmay then be packed in membrane bound vesicles. Withoutexposure to MSH or cAMP, or in the absence of some otherstimulation, e.g., choleratoxin (16) or cell-to-cell interactions(7), tyrosinase would remain inactive either as a protyrosi-nase (17) or as an inhibited enzyme (18), or perhaps both.Still in the preparatory phase, MSH receptors could be syn-thesized and provided with a carbohydrate moiety in theGolgi complex. The receptors may then reach the cell sur-face (12) at the end of the S phase of the cell cycle or appearon the surface earlier in a masked form. "Unmasking"would then take place in G-2. Our recent findings on the ef-fect of neuraminidase on the display of MSH receptors fa-vors the latter interpretation (Fritsch and Varga, in prepara-tion). By the time that active MSH receptors, premelano-somes, and inactive tyrosinase are formed, the preparatoryphase of pigmentation would be completed.The binding of MSH to melanoma cells (2) initiates a

chain of events, the endproduct of which is the melanizedmelanosome (14). One of the earliest events is an accumula-tion of cAMP, which takes place within minutes after MSHis bound to the cells. The accumulation of cAMP is transito-ry; it is maximal in 30 min and drops to the unstimulatedlevel in about 2 hr (4). For this reason, we can assume thatcAMP induced by MSH acts as a short-term signal only.After the rise in the concentration of cAMP, another reac-tion must occur that in turn would control the events thatfollow. These events, between the synthesis of cAMP and theemergence of active tyrosinase, are obscure. One step couldbe the activation of a cAMP dependent protein kinase (19),and another step the internalization of the MSH-receptor-adenylate cyclase complex, that brings cAMP close to thecenters of melanization. De novo synthesis of tyrosinaseprobably does not take place because the process is notblocked by inhibitors of protein synthesis (20).

Because the cytochemical localization of the dopa reac-tion in aldehyde-fixed cells indicates the presence of tyrosi-nase in the Golgi-associated vesicles and smooth membra-nous channels that link the Golgi zone with melanosomes

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562 Cell Biology: Varga et al.

(10), it is possible that the activation step in vivo is related tothe transfer of tyrosinase from the Golgi complex to the me-lanosomes. Golgi cisternae and vesicles do not get melanizedin vivo, but the channels sometimes do (Moellmann, unpub-lished observation on frog melanocytes). It is conceivablethat melanization of the vesicles is prevented by a perme-

ability barrier (21) to tyrosine or by the presence of a tyrosi-nase inhibitor. Melanization may be initiated by the fusionof surface-derived vesicles, containing the MSH receptor-activated adenylate cyclase complex, with the smooth sur-

faced vesicles that contain inactive tyrosinase and with pre-

melanosomes (22), which are permeable to L-tyrosine or

which contain a tyrosinase activator.Our demonstration of the internalization of FITC-MSH

may suggest a biological role for hormone internalization.One prediction of this model is that MSH-induced cAMPdoes not diffuse freely within the cytoplasm, but that it is lo-calized, together with the MSH-receptor-adenylate cyclasecomplex, in the Golgi region. Our recent observations on theGolgi-related accumulation of cAMP, demonstrated by an

immunocytochemical technique, strongly supports the valid-ity of this prediction (in preparation). Cyclic AMP may acti-vate a kinase in the proximity of its formation and may bedegraded soon after. Phosphodiesterase in chick embryo fi-broblasts is located at the plasma membrane (23). An in-triguing question is the long delay between the appearance

of hormonally induced cAMP and activation of tyrosinase. Itis possible that this delay is related to the process of internal-ization and fusion of vesicles.

In summary, we have provided evidence that indicatesthat the initial events in the execution of MSH action are

confined to the Golgi region. Earlier observations haveshown that formation of premelanosomes and activation oftyrosinase occur in the same region. These findings taken to-gether favor the hypothesis that instructions resulting fromthe attachment of MSH to its receptors in mouse melanomacells are carried out in a compartmentalized manner andthat this process includes the Golgi complex.

We thank Dr. George Palade and Dr. Alex Novikoff for theirhelpful suggestions concerning the manuscript. This study was sup-

ported by USPHS Grants CA-15557 and CA-04679-16, American

Cancer Society Grant BC3I, and by a Max Kade Fellowship to Dr.Peter Fritsch.

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