Localization of lh receptors on luteal cells with a ferritin-lh conjugate

Molecular and CellularEndocrinology, 15 (1979) 61-78 0 ElsevierjNorth-Holland

61

Scientific Publishers, Ltd.

LOCALIZATION OF LH RECEPTORS ON LUTEAL CELLS WITH A FERRITE’&LH CONJUGATE J.L. LUBORSKY and Harold R. BERMAN Re~r~d~ct~veB~olo~ Section, ~e~art~e~t of Obstetricsand Gynecology, Yale University School of medicine, New Haven, CT 06510 (U.S.A.] Received 5 April 1979; accepted 22 May 1979

In order to study the distribution of LH (HCG) receptors on luteal cells ferritin was coupled to ovine LH with glutaraldehyde and purified by gel chromato~aphy. The conjugate CFELH) competed with r“1-hCG for binding to isolated luteal membranes and stimulated a dosedependent release of progesterone (P) from isolated luteal cells which was inhibited by PGFr,. FELH was distributed as single molecules or in small clusters at intervals on the surfaces of luteal cells labeled at 37”C, 4°C or with formaldehyde preflxation. Capping or preferential labeling at one site was not observed. The general distribution of LH (hCG) binding sites at 37*C was confirmed by light-microscopic autoradiography. The distribution at 4’C or with prefixation was more diffuse than at 37°C suggesting that FELH binding induces small changes in receptor aggregation. Binding of FELH was specific since excess hCG reduced FELH binding to luteal cells. In cells labeled at 4”C, rinsed and warmed to 37°C FELH was observed along cell surfaces and within some coated vesicles and a few lysosomes within minutes suggesting that receptor internalization is a rapid and possibly continual process. Keywords: receptor distribution; zation.

cell membrane; luteinizing hormone; ovary; internali-

The spa&l arrangement of receptor on the cell surface may be an ~portant element in the expression of hormone action. The distribution of cell-surface recep tors has now been described for a variety of receptors (Albertini and Anderson, 1977; Anderson et al., 1976; Hopkins and Gregory, 1977; Jarett and Smith, 1975; Kahn, 1976; Ko et al., 1977; Nelson et al., 1978; Orci et al., 1975; Varga et al., 1976a, b). In general, each cell system appears to exhibit different but characteristic receptor distributions. Receptors in some systems have a relatively unrestricted, patchy distribution along cell surfaces, such as those for insulin on adipocytes (Jarett and Smith, 1975), LHRH on gonadotrophs (Hopkins and Gregory, 1977) and epidermal growth factor on fibroblasts (Schlessinger et al., 1978). Other receptors have a restricted distribution on the cell surface which may be associated with a particular cellular structure such as those for low-density lipoprotein (LDL) which are found in “coated pits” on the surface of fibroblasts (Anderson et al., 1976), melanocyte-stimulating hormone (h4SH) receptors which have a polar location asso-

62

J.L. Luborsky, bails

R. Behrrnan

ciated with the Golgi apparatus on melanoma cells (Varga et al., 1976a, b) and those for insulin on placental trophoblasts which are found only on the microvilli (Nelson et al., 1978). Thus receptor distribution may be related to the specific function of the hormone-receptor complex and/or to the functional state of the cell. In addition to a redistribution of receptors on the cell surface many types of receptors appear to be redistributed to intracellular sites following hormone binding (~bertini and Anderson, 1977; Anderson et al., 1976; Carpenter and Cohen, 1976; Chen et al., 197’7; Conn et al., 1978; Das and Fox, 1978; Goldfine et al., 1977; Gordon et al., 1978; Moelfmann et al., 1978; Schlessinger et al., 1978; Varga et al., 1976b; Willingham et al., 1978). In some systems receptors undergo lateral aggregation to form large patches which are subsequently internalized (Hopkins and Gregory, 1977; Schlessinger et al., 1978). In other systems, particularly those in which the receptors have a restricted distribution, the receptors are internalized without further apparent redistribution on the cell surface (Anderson et al., 1976; Varga et al., 1976b). Internalization of labeled binding sites to specific organelles such as lysosomes (Anderson et al., 1976; Carpenter and Cohen, l976; Chen et al., 1977), premelanosomes ~oellmann et al., 1978), nuclei (Goldfme et al., 1977) and the Go&i apparatus (Albertini and Anderson, 1977; Farquhar, 1978) has been reported. Internalization is required for LDL usage since the free cholesterol obtained by lysosomal enzyme action is used to regulate steroid metabolism in fibroblasts (Goldstein and Brown, 1977). However, the relationship of internalization and cellular function has not been clearly established for many other systems. Luteinizing hormone (LH) is a glycoprotein hormone which promotes ovulation and luteinization in the ovary and stimulates progesterone production in luteal cells. Initially, LH binds with high affmity and specificity (Ryan and Lee, 1976) to cell-surface receptors on luteal cells (Han et al., 1974; Rajaniemi and VanhaPerttula, 1972; Rajaniemi et al., 1974; Richards and Midgley, 1976) and activates adenylate cyclase by an as yet undefined mechanism (Marsh, 1976). The activation of adenylate cyclase results in the intracellular accumulation of cyclic AMP. Either LH, human chorionic gonadotropin (hCG) (which also has a high specificity for the LH receptor) or exogenous CAMPcan stimulate steroidogenesis and the subsequent secretion of progesterone by luteal cells (Marsh, 1976). Recent studies have shown that LH may regulate its own receptor (Harwood et al., 1978); LH receptor binding and adenylate cydase activity decrease within 24 h in vivo, in response to a pulse of hCG. Part of this long-term effect of hCG appears to involve an internalization of hormone-receptor complexes (Conn et al., 1978) to lysosomes (Chen et al., 1977). There have been few studies of LH receptor distribution. LH receptors were shown to move laterally in the plane of the ovarian cell membrane to form large aggregates or “caps” when labeled by an indirect procedure that used fluorescein labeled LH antibodies (Amsterdam et al., 1977). This suggests that these receptors could undergo specific redistribution in response to LH binding or to the action of other regulatory hormones. However, antibody binding mai itself elicit a receptor redis-

LH receptor distribution on luteal eelis

63

tribution that is not necessarily related to hormone binding or to cell function. In order to extend present knowledge of the function of the LH receptor, we have developed and partially characterized a ferritin-LH conjugate (FELH) to examine the distribution of LH receptors at the ultrastructural level on isolated luteal cells. The primary objective of these studies was to establish the general distribution of LH receptors on isolated luteal cells and to assess its possible relationship to LH binding. In addition, FELH intern~ization was examined as part of preliminary studies to determine the significance of receptor internalization in relation to cell function and hormone action. Early results of this study were reported in abstract form (Luborsky-Moore and Behrman, 1978).

MATERIALS AND METHODS Animzls. Immature, 26day-old rats (CD strain, Charles Rivers Laboratories, Wilm~gton, MA) were injected with 50 IU (SC) of Pregnant Mares Serum (Gestyl, Organon) and 64 h later with 25 IU (SC)of human chorionic gonadotrop~ (A.P.L., Ayerst). This treatment synchronized ovulation and maximized corpus luteum formation. Luteal cell isolation. Ovaries were removed 7 or 8 days after hCG injection. Luteal cells were dispersed by a modification of methods described previously (Thomas et al., 1978; Wilkinson et al., 1976). Minced tissue was incubated in low calcium miniial essential medium (MEM) (Gibco, No. 138) with collagenase and DNAase Worthington Biochemical Co., NY) (1 h, 37*C) under 95% 0zf5% COZ in a siliconized 25ml erlenmeyer flask. The tissue was then centrifuged (50 Xg, 5 min), resuspended in low calcium MEM containing 3 mM EDTA, gently drawn in and out of a Pasteur pipette (pipetted) about 10 times and incubated for 2 min at 22°C. The suspension was subsequently centrifuged (50 Xg, 5 min) and resuspended in MEM containing calcium (GIBCO, No. 236 or No. 199). The tissue pieces were pipetted gently, the clumps allowed to settle (0.5-l min) and the “supernatant” fraction containing isolated cells removed and faltered through nylon mesh (Nyten, Tetko Inc.). Fresh MEM was added to the cell “pellet” and the procedure repeated until the clumps were dispersed. Thus, isolated cells were sub. jetted to a minimum of mechanical ma~pulation. Following isolation, cells were preincubated in MEM without treatment (90 min, 37’C, 95% 0~~5% CO%)before use. Cell concentration was determined with a hemocytometer and cell viability by trypan blue exclusion (Thomas et al., 1978). Typically, a population of 70-90% luteal cells was obtained based on cell size and characteristic lipid inclusions CrJilkinson et al., 1976); cell viability was 80-90% of the total cell population before and after each experiment.

Ferritin-LH conjugation. The FELH conjugate was prepared by modifying previously described methods (Avrameas, 1969; Jarett and Smith, 1975). LH

64

J.L. Luborsky, Hmold R. Behrman

(5 mg, NIH-LHSIB) was dissolved in 1 ml of potassium phosphate buffer (0.2 M, pH 6.8) and 2 ml of 100 mg/ml of ferritin (EM grade, Polysciences) was added. Glutar~dehyde (0.28 ml of a 1% solution in distilled water) was added slowly with continuous mixing. The final reaction mixture (5 mg LH, 200 mg ferritin, 0.061 M potassium phosphate buffer, 0.085% glutar~dehyde, pH 6.8) was incubated for 90 min at 22’C with continuous mixing. The conjugation solution was dialyzed against 0.1 M sodium-O.01 M potassium phosphate buffer, pH 6.8, for 24 h at 8°C to remove glutaraldehyde. To separate unconjugated LH from FELH, 12’1-oLH or “‘1-hCG was added and the conjugation solution immediately applied to a Sephadex G200 or Biogel A5m colwnn preequilibrated with 0.2% BSA and eluted with sodium-potassium phosphate buffer (pH 7.3) containing 0.2% BSA. Fractions (1 ml) were collected and the absorbance (440 nm; ferritin) and radioactivity (*251-LH or r2’I-hCG) determined. FELH was readily separated from unconjugated LH. Fractions with ferritin but no free LH were pooled and concentrated by ultrafiltration (AMICON XMIOOA filter). FELH was analyzed for LH-receptor binding activity with rat luteal membranes and for biological activity by their ability to stimulate progesterone production in isolated luteal cells. Concentrations of FELH are expressed in terms of equiv~ent hCG activity as determined in the binding assay. evocation of FELIX To remove unconjugated ferritin and inactive FELH, FELH (250 ng) was incubated with 200 mg of isolated luteal membranes in 2 ml of 0.04 M Tris-HCl -I-0.2% BSA (pH 7.4) for 16 h at 22*C. The membranes were rinsed in the same buffer (3X), centrifuged (2000 X g, 10 min) and the pellet resuspended in 1 ml of MEM. The suspension was placed in a 60°C water bath for 10 min to release bound FELH (Behrman and Hichens, 1976), centrifuged (2000 X g, 10 min) and the supernatant containing purified FELH (pFELH) retained. About 45% of the LH-receptor binding activity of FELH was recovered. Membrane-binding assay. A procedure for quantitating LH receptors based on the use of plasma membranes from homogenates of luteinized ovaries was used (Hichens et al., 19’74). Quantification of LH, hCG or their conjugates was assessed by competition with “‘1-hCG for membrane binding and compared to that obtained with known concentrations of purified hCG. Iodination of hCG was performed with lactoperoxidase (Hichens et al., 1974). Rad~oimmunoussay. Follow~g incubation of cells with free or conjugated hormones, cells were centrifuged (5 min, 100 X g) and the media removed for progesterone radioimmunoassay (Orczyk et al., 1979). Electron microscopy. Cells were fixed in 2% glutaraldehyde in cacodylate buffer (pH 7.3) with 1% tannic acid (320 mOsM, 1 h, 22”(Z), rinsed in cacodylate buffer with sucrose (310 mOsM, pH 7.3), postfixed in 2% 0~04 (1 h) and pelleted.

LH receptordistributionon Meal ceils

65

Pelleted cells were dehydrated and embedded in Epon 812. Thin sections were obtained with a Reichert OMU2 ultr~crotome and viewed without staining in an Hitachi 12 electron microscope, In some exper~ents, cells were prefixed in 0.1% f~rm~dehyde in phosphate-buffered saline (PBS) for 2 min and rinsed extensively in 0.25 pgtgfmlsodium borohydride in PBS (310 mOsM) to block unreacted aldehydes, prior to incubation with FELH (Hopkins and Gregory, 1977). Autoradiography. Isolated cells were incubated in 12 X 75 glass tubes with 12’I-hCG (1.2 X 1O6 cpm; 190 000 cpm/ng) alone or with unlabeled hCG (100 IV/ ml) in MEM at 37°C for 90 min. The cells were then rinsed in fresh media with added hormones but without 12’I-hCG and aliquots attached to glass slides for 20 min at 37°C. Cells were fured in 2% glutaraldehyde as above (30 min, 22’C, pH 7.3), rinsed in buffer and airdried. The slides were dipped in Ilford K2 emulsion (PolySciences Inc.), exposed for 10 days and developed in Microdol. Slides were stained with hematoxylin/eosin, observed in a Zeiss Photomicroscope I and photographed with Kodak P&s-X ftlm. ~soe~ect~c focusing. To determine the net charge of the FELH, the isoelectric point(s) were determined with an LKB Model 8101 column (110 ml capacity) on a pH gradient from 3 to 10. A linear sucrose gradient up to 43% containing the ampholyte (LKB 3110) was applied to the column with an LKB 8 121 gradient mixer and the sample was applied to the middle of the gradient. Electrofocusing was carried out at 8°C for 20 h. An initial potential of 500 V was maintained for 2 h and then increased to 1000 V. Fractions (2 ml) were collected and assessed for ferritin absorbance and for radioactivity. Experimental procedure. Cells (104/ml) were incubated in MEM (1 ml) in 12 X 75 glass test tubes for 90 min (15,24) with 95% 0,/S% COz. They received the following treatments: (1) FELH at 37”C, 4°C or with prefixed cells at 37’C or 22’C. (2) FELH t hCG (100 IV/ml) at 37°C (to determine nonspecific binding). (3) FELH + PGF;?, (1 pg/ml) at 37°C. (4) Free ferritin (0.5 mg/ml) at 37*C. (5) LH. (6) PGFza (1 &ml). (7) LH t PGF2,. (8) No treatment. At the end of incubation cells were centrifuged (100 Xg, 5 min), the media was assayed for progesterone and the pellet was rinsed and futed for electron microscopy. To examine FELH intern~ization cells were incubated at 4’C for 90 min with FELIi (100 ng/ ml) or purified FELI-I (70 &ml), rinsed at 4°C and post-incubated for 2 min, 30 min or 120 min at 37*C and fixed for electron microscopy.

RESULTS Properties of the ferrOin-I.3 conjugate The distribution of ferritin and LH in fractions obtained by chromatography of

J.L. Luborsky,

66

Harold R. Behrman

eiOgeiii5m ?s*

20

2000 t Ii 1500 J 0N

IO00

ZE Fk 500

0

4 I

20

30

40

50

60

70

80

90

FRACTION

Fig. 1. Chromatography of FELH on Biogel ASm. Fractions were assessed for radioactivity due to free hormone (0) and for ferritin absorbance at 440 nm (0). Fractions 2.5-4.5 were pooled, concentrated and tested for the presence of free hormone, hormone binding and biological activity (see text). Similar results were obtained with Sephadex G200 except that FELH was eluted in a smaller volume.

the conjugation solution with “‘1-LH added just prior to ~llromatography is shown in Fig. 1. Fractions (tubes 25-45) containing ferritin but no free LH (“‘I-LH) were pooled (FELH) and concentrated, The first few fractions exhibiting ferritin absorbance frequently contained ferritin aggregates and were omitted from the pool. Chromatography of unconjugated ferritin and “‘1-LH on the same column produced two single peaks similar to that seen in Fig. 1; one due to ferritin and the other to LH (data not shown). When ‘251-hCG(LH) was included during conjugation, radioactivity was found in all ferritincontaining fractions as well as in fractions containing free hormone (data about 25% of the hormone was connot shown). Based on ‘251-hCG incorporation, jugated to ferritin (i.e., appeared in the ferritin peak). The fractions pooled for use as FELH contained 13% of the original LH (710 pg) and 40% of the original ferritin (80 mg) in a final volume of 4.5 ml. Thus the molar ratio of total (free and conjugated) ferritin : LH was approximately 3.3. LH(hCG) binding activity in the FELH was usually 1-2 pg/ml or 1.2% of the total incorporated LH. Less than 0.5 r&ml of free hormone (LH(hCG) binding activity) was detected in the supernatant following sedimentation of FELH by centr~fugation at 65 000 X & for 2 h at 4°C. The isoelectric point of the FELH was about pH 5 similar to that of the unconjugated ferritin in the crude conjugate and the native ferritin (pH 4.6). The FELH retained 80% of its binding and biological activity during storage for 2 months at 4°C. The concentration of LH binding activity in the conjugate was determined by assay and competition with ‘251-hCG in the isolated ovarian membrane-binding used at appropriate dilutions to test the ability of FELH to stimulate progesterone

61

LH receptor distribution on luteal cells

(L

a

0

25

50

EQUIVALENT

100

FE

25

hCG ACTIVITY

50

100

(ng/ml)

Fig. 2. The biological activity of FELH was secretion of progesterone from isolated luteal in an LH-receptor binding assay and expressed in triplicate for 90 mm at 37°C with (A) LH terone. Ferritin alone did not significantly expressed as mean f S.E.

shown by its ability to elicit a dose-dependent cells. The concentration of FELH was estimated as equivalent hCG activity. Cells were incubated or (B) FELH and the media assayed for progesstimulate progesterone secretion. Values are

secretion from luteal cells. Thus all data are expressed in terms of equivalent hCG activity. Since this assay is based on competition with “‘1-hCG the results also showed that FELH could compete with hCG for binding to the LH receptor. Cells incubated with FELH responded with a dose-dependent increase in progesterone secretion (Fig. 2). The shape of the dose-response curves for LH and FELH was similar. Autoradiography of ’ ’ 'I-hCG labeled lu teal cells Cells labeled with 1251-hCG appeared uniformly labeled (Fig. 3). Grains were apparent over most luteal cells 090%) in varying amounts. No large patches or caps were seen. It was not possible to determine whether the radioactive source was on the cell surface or if it was internalized. Cells incubated with 12’I-hCG and an excess of unlabeled hCG to examine nonspecific binding were generally unlabeled. A few (
68

Fig. 3. Autoradiography of 1251-hCG bound to isolated rat luteal cells at 37°C. (A, B) 12%hCG (190 000 cpm/ng, 6 ng). (C) 12SI-hCG and excess hCG (100 IU/mI) (see text for details). Cells in A, B and C were processed identically except for hematoxylin/eosin staining. Cells typically had grains distributed over the entire cell (A, B) while no grains were apparent over control (C). (A, B) 1400x. (C) 1800x. Fig. 4. Luteal c&i incubated with FELH (100 ng/ml) at 37’C for 90 min. Ferritin molecules (v) are typically distributed at irregular intervals along the cell surface either as single moiecules or more frequently in clusters. Ferritin is also seen in iysosome-like (*I structures within cells. Unstained section. 76 000X.

Fig, 5. Luteal cell incubated with FELH (SO ng/ml) at 37°C for 90 min. Ferritin (~1 was distributed as in Fig. 4 on many ceils but there was also a more frequent occurrence of surfaces with single or double molecules and no clusters. Unstained section. 76 000X. Fig. 6. Control luteal cell incubated with FELR (100 ngfml) and excess hCG (100 IUfmlI at 37’C have few ferritin cores (r) on their free surfaces. Unstained section. 71 000X.

J.L. Luborsky, Harold R. Behrman

Fig. 7. Lute& cells incubated with FELH (100 n~ml} at 4*C for 90 min. Ferritin moIecules (4 are dis~~buted atong the cell surface, p~dom~an~y as single molecules. FELH did not appear to be preferent~a~y located on cellular extensions (A) or in coated regions or “pits” (‘II) (A, B) on the membrane. Some clusters were observed (A, C). Unstained sections. 76 000X.

LH receptor distribution on lute& cells

11

single molecules interspersed between the clusters, or occasionally in small patches of lo-20 molecules, in close proximity to the cell membrane. Also, FELH did not appear to be preferentially localized on membrane extensions such as surface folds or ruffles or in coated pits. At lower concentrations of FELH, ferritin was distributed similarly but fewer clusters of molecules were seen. An example of a cell surface without FELH clusters is shown in Fig. 5. FELH binding was specific since luteal cells incubated in FELII and an excess of hCG (Fig. 6) at 37°C had few ferritin cores on their free surfaces. Some single molecules and small aggregates were seen in intercellular spaces and between closely apposed surface projections. Similarly, ferritin alone (0.5 mg/ml) did not bind to luteal cells although it was found in lysosome-like structures after incubation of cells at 37°C. Specific binding to erythrocytes and follicular cells (i.e., those with little cytoplasm, no smooth endoplasmic reticulum or lipid droplets) was not observed. Cells were also labeled at 4°C (Fig. 7) or after prefixation with formaldehyde to determine if the FELH distribution was changed by reducing membrane fluidity. Generally, the pattern of ferritin molecules was similar to that at 37’C in that ferritin was found on all parts of the cell surface as single molecules, small clusters and a few patches. However, single FELH molecules, rather than clusters, predominated. Preferential labeling with FELH in association with membrane thickenings was not seen ~thou~ such structures were evident along the membrane. The total number of receptors (FELH-boding sites) per cell was estimated from counts of the number of ferritin molecules (12 + 5) per micron of membrane surface and the approximate section thickness (0.08 pm). Cells incubated in 100 ngf ml FELH were used for this purpose. The average diameter of the isolated rat luteal cell was 10 pm. There were about 5 X lo4 receptors/cell at 37°C.

To examine the possible ~terna~~tion of FELH, cells were labeled in several ways. In some experiments, cells were labeled with FELH (IO0 n&ml) for 90 min at 4’C (2 min) and postincubated at 37°C for 2 min, 30 min or 2 h. In other experiments FELH was further purified to remove unconjugated ferritin (pFELH: described in the methods section). The pFELH retained biological activity (i.e., competed with “‘1-hCG in the membrane-binding assay and stimulated progesterone secretion from isolated luteal cells). Cells labeled with pFELH at 4’C were rinsed and post~cubated for 2 h at 37°C. The result of labeling with FELH (Fig. 8) or pFELH at 4’C and subsequently warming to 37°C was similar. Ferritin was still distributed at intervals along luteal cell surfaces. In addition, some cells contained ferritin in small coated vesicles (Fig. 8) and occasionally in 1 or 2 lysosome-like structures (Fig. 9). The ferritin seen in the small coated vesicles appeared to be bound to the membrane rather than free within the vesicle since the molecules were spaced evenly along the inner surface of the coated membrane. Few cells (2%) fixed at 2 min after labeling with

72

J.L. Luborsky, Harold R. Bt

. ,_ ,

LH receptor ~~~tribut~onon luteal cells

73

FELH at 4OC had ferritin in lysosomes. No difference was observed between cells postincubated at 37°C for 30 min or 2 h, except that a larger number of cells appeared to contain ferritin particles. Cells incubated with FELH or pFELH at 37*C also contained ferritin particles in vacuoles that did not appear to be bound to the vacuolar membranes (Fig. 8; inset) and thus were probably endocytosed nonspecifically.

DISCUSSION Properties

of the FELH

This study shows that LH, a glycoprotein hormone which consists of two noncovalently linked subunits, can be conjugated to ferritin with sufficient retention of the binding and biological activity of the LH for use of the conjugate in physiological and ultrastructural studies. The FELH probably contains both the cyand /3 subunits of LH since either alone are not biologically active (Sairam and Papkoff, 1974). Evidence that the FELH had LH activity similar to unconjugated LH was shown in two ways. First, FELH stimulated progesterone secretion with a doseresponse relations:+ that was similar to that of free LH. Second, FELH-stimulated progesterone secretion was consistently inhibited by PGFzo, which also inhibits LHstimulated progesterone secretion without a reduction of bound LH (Behrman, 1979; Lahav et al., 1976; Thomas et al., 1978). In addition, the degree of in~bition by PGFzo, was similar irrespective of whether or not the LH was conjugated to ferritin. Although similar yields of active ferritin conjugate were obtained for other hormones (Jarett and Smith, 1975; DiPasquale et al., 1978), consideration of the final yield of biologically active FELH suggested that its LH activity (1.3%) could arise from unconjugated (noncovalently bound) LH. However, less than 0.27 ng/ml or 0.02% of the LH activity was found in the supernatant following centrifugation (to separate FELH and free LH) of FELH suggesting that the majority of active LH was linked to ferritin. In addition, in preliminary experiments FELH was bound to a Con A-Sepharose column which binds LH but not ferritin alone. When the bound material was eluted with ~-methylmannoside or 1 M NaCl ferritin and LH were

Fig. 8. Intracellular distribution of FELH following incubation of luteai cells with FELH at 4“C, and postincubation for 30 min at 37°C. Some ferritin (rf molecules remain bound to the surface while others are bound to coated regions (vv) of internalized membrane. Unstained section. 76 000X. Inset: Nonspecific internalization via endocytosis may also occur when cells are labeled at 37’C, but in these instances ferritin does not appear to be bound to the vacuole membrane. Unstained section. 72 000X. Fig. 9. Ferritin (v) in lysosome-like structures within luteal cells following labeling, rinsing at 4°C and postincubation of cells at 37°C for 30 mm. Unstained section. 76 000X.

74

J.L. L&xx-sky, Harold R. Behrman

found in the same fractions indicating free LH was not present in the conjugate (Luborsky-Moore, unpublished observation). The monomeric nature (see below) of the FELH and its specificity in relation to other competing hormones has yet to be determined. The specificity of this conjugate was shown by the competitive inhibition of its binding to luteal cells by excess hCG (an LH analog produced by the human placenta). We have not yet established whether the conjugate is primarily monomeric with respect to ferritin. If the majority of ferritin particles have one or more LH molecules per ferritin, the degree of clustering and patching would be temperaturedependent. On the other hand, if there were greater than one ferritin per LH molecule the degree of clustering would be temperature-independent. Since the occurrence of single ferritin molecules increased substantially at 4OC,the FELH probably contains relatively little ferritin-ferritin aggregates. Topographical distribution

In this study we have shown that LH receptors have an unrestricted distribution on isolated luteal cells in agreement with an earlier study with ferritin-hCG reported in abstract form (Rajaniemi and Jaaskelainen, 1976). In general, FELH was uniformly distributed and consisted of single molecules and small, variable sized patches at intervals along the cell surface similar to receptors for insulin on adipocytes (Jarett and Smith, 1975) and on liver cells (Orci et al., 1975), epidermal growth factor on fibroblasts (Schlessinger et al., 1978) and LHRH receptors on gonadotrophs Hopkins and Gregory, 1977). FELH binding did appear to induce small changes in the grouping of LH receptors since their distribution was more diffuse when labeled at 4°C. However, FELH binding did not elicit the formation of “caps” (a massive aggregation of receptofs at a single site on the cell surface). Thus, capping does not appear to play a role in LH-receptor function as suggested by an earlier study (Amsterdam et al., 1977). Small changes in the grouping of receptors may be required for hormone action. Intact antibodies to the insulin receptor which are capable of cross-linking recep tors, were recently shown to mimic the action of insulin while the monovalent FAB fragments did not (Kahn et al., 1978). Similarly, when basal glucose transport was inhibited by c~ochalasin, and insulin receptors were labeled with ferritin-insulin, the number of ferritins~~oup (patch) was decreased (Smith and Jarett, 1977). Preliminary quantitative analysis of FELH on cells labeled at 37°C or 4°C or in the presence of PGF,, suggests that changes in the grouping of FELH molecules may occur with changes in the functional state of luteal cells (Luborsky-Moore, unpublished observations). The estimated number of receptors/cell (5 X 104) is in agreement with data obtained by Scatchard analysis of hCG binding to luteal cells (105) (Luborsky-Moore, unpublished observations; Papaionannou and Gospodarowicz, 1975) and is similar to that for other peptide and polypeptide hormone receptors (Hopkins and Gregory, 1977; Kahn, 1976).

LH receptor distributionon lutealcells

15

In contrast to the results obtained in this study, the distribution of LH receptors seen on intact luteal tissue by EM autoradiography of ‘2sI-hCG injected in vivo suggests that LI-I receptors. are more numerous on the microvilli and folded regions of luteal cell membranes adjacent to the vascular spaces (Han et al., 1974). This restricted distribution of LH receptors in vivo could be due to the extensive intercellular junctions between luteal cells (Albertini and Anderson, 1975). The junction would limit penetration of the 12sI-hCG and/or define restricted membrane “domains”. These restrictions would not be present on isolated luteal cells. However, unrestricted receptor distributions are not necessarily characteristic of isolated cells since a restricted distribution of receptors for LDL is observed on fibroblasts (Anderson et al., 1976) and for MSH on melanoma cells (Varga et al., 1976a, b) in vitro. Further experiments directed at a comparison of LH receptors on intact tissue and on isolated cells are required before specific conclusions can be drawn.

There is accumulating evidence that prolonged interaction of the hormone and its receptor results in an eventual reduction of the receptors on the cell surface in many systems (Harwood et al., 1978; Kahn, 1976; Schlessinger et al., 1978; Varga et al., 1976b). Studies of the LH receptor have shown that physiological doses of LH or hCG can induce desensitization and eventual receptor loss (Harwood et al., 1978). One possible mechanism of receptor loss in addition to shedding or enzyme inactivation is by internalization (Kahn, 1976). It has been shown that the internalized LH remains bound to the receptor (Conn et al., 1978) and that the radioactivity due to hCG is found in lysosomes (Chen et al., 1977). The time periods examined for this internalization in most studies were greater than 3 h (Conn et al., 1978; Chen et al., 1977). However, the results of this study suggest that internalization is an ongoing process that can be seen within minutes at 37°C as has been

Table 1 Effect of PGF2, on FELH- or L&induced

progesterone

Treatment

Progesterone (ng/104ceUs)

0 LH

8.3 20.6 12.6 13.8 22.6 14.4

PGF2,

LH t PGF,, FELH FELH + PGF, cy

secretion

f 0.9 f 1.0 i 0.6 I: 1.4 f 1.4 + 0.6

Inhibition of FELH- and LH&imulated progesterone secretion from isolated luteal cells by PGFz.. Cells were preincubated in PGF2,(1 &ml) for 30 min at 37”C!, FELH or LH (50 ng/ml; concentration refers to equivalent hCG-binding activity) added and the cells further incubated for 90 min at 3r”C. Values represent mean f: SEM of each treatment performed in triplicate.

76

J.L. Luborsky, Ha&d R. Behrman

shown for the receptor for epidermal growth factor (Das and Fox, 1978). It was also possible to show what appears to be FELH bound to membranes of coated vesicles suggesting that as in other systems (Anderson et al., 1976; Moellmann et al., 1978; Willingham et al., 1978) the hormone-receptor complex may be removed from the cell surface via these structures. Although internalization has been shown to occur in this and other studies its significance in relation to luteal cell function has not been established. The results of this study show that LH receptor internalization is rapid, unlike the loss of receptors following desensitization (Harwood et al., 1978). Thus, internalization may be a continual process that is simply a function of receptor turnover and is only indirectly or secondarily related to the loss of receptor function. Further, more detailed studies of this process are in progress. It was recently reported that binding and internalization may be effected by the overall charge of the ligand (Farquhar, 1978). Native ferritin (pl= 4.6) did not bind to pituitary cell surfaces but was taken up by endocytosis and concentrated in lysosomes; cationic ferritin (‘pI=,8) did bind to pituitary cells and was found in Golgi elements, secretory granules and lysosomes. Thus the total charge of a molecule may determine its route upon internaiization. The FELH used in this study had a p1 of about 5 which differs from that of LH or hCG (plz7-8). However, internalized LH (hCG), whether labeled with **‘I (Chen et al., 1977) or with ferritin has never been observed elsewhere than in presumed lysosomes of luteal cells. Thus, the LH was probably not directed to unusual sites by the presence of ferritin. In conclusion, the results of this study suggest that LH receptors are distributed randomly on the surface of isolated luteal cells. Upon binding of FELH, the receptors cluster, an event which may be required for the expression of LH action. In ad~tion, some but not all the FELH bound to the cell surface is rapidly internalized .

This work was supported by an AAUWpostdoctoral fellowship (1977-1978),NIH postdoctoral fellowship IF32 NS05974 (197%1979), NIH Grant HD-10718 and Ford Foundation Grant 770-0534. PGF2, was a gift of Dr. John Pike, The Upjohn Co. (Kalamazoo, MI) and purified hCG was a gift of Dr. M. Hichens, The Merck Institute (Westpoint, PA). The authors would like to thank Dr. A. Eisenfeld and Dr. J. Jamieson for valuable discussions and S. Losacco for typing the manuscript.

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