Peptides.Vol. 11, pp. 737-745. ©Pergamon Press plc, 1990. Printed in the U.S.A.

0196-9781/90 $3.00 + .00

Characterization of the Detergent Solubilized Receptor for Gastrin-Releasing Peptide D. C I R I L L O , *1"2 L. N A L D I N I , .1'2 T. W. M O O D Y , ? P. COMOGLIO,:I: J. S C H L E S S I N G E R .3 A N D R. K R I S .4

*Rorer Biotechnology, Inc., 680 Allendale Road, King of Prussia, PA 19406 "PDepartment of Biochemistry, The George Washington Medical Center, Washington, DC ¢Department of Biomedical Sciences and Oncology, University of Torino Medical School, Torino, Italy R e c e i v e d 5 February 1990

CIRILLO, D., L. NALDINI, T. W. MOODY, P. COMOGLIO, J. SCHLESSINGER AND R. KRIS. Characterizationof the detergent solubilized receptorfor gastrin-releasingpeptide. PEPTIDES 11(4) 737-745, 1990.--Properties of detergent solubilized gastrin-releasing peptide receptor were investigated. Swiss 3T3 membranes were covalently labeled with [~25I]GRP and homobifunctional cross-linkers. A major labeled protein of 75 kDa was resolved using SDS-polyacrylamide gel electrophoresis. When the same preparation was solubilized with zwitterionic detergent and analyzed under nondenaturing conditions the protein bound radioactivity was resolved in two different peaks, a major one of apparent molecular weight 220,000 (peak 1) and a minor one of 80,000 (peak 2) both containing the 75 kDa protein. Specific ligand binding activity also eluted with peak 1. These results indicate that the active form of bombesin/GRP receptor is a large complex containing the 75 kDa ligand binding domain. Receptor

Neuropeptide

Peptide

Solubilization

Gastrin-releasing peptide

3T3 cell lines has been attributed to differential glycosylation. The molecular weight of the core protein was shown to be approximately 45 kDa (13). Because these studies utilized denaturing detergent (SDS), the occurrence of a more complex structure, held together by noncovalent interactions, could not be evaluated. We have previously found that the bombesin/GRP receptor can be solubilized with functional binding properties by the combination of the zwitterionic detergent CHAPS and the sterol ester cholesteryl hemisuccinate (CHS) (21). Here the receptor was covalently labeled in Swiss 3T3 membranes with [125I]GRP and the cross-linker EGS, solubilized and analyzed under nondenaturing conditions by molecular sieving chromatography and sucrose density gradient sedimentation. A high and a low molecular weight form of the receptor resulted. When a soluble receptor assay was performed on fractions collected from chromatography of detergent-solubilized membranes, specific binding activity for radiolabled ligand eluted with the same volume as the high molecular weight form. Our data suggest that the active form of the bombesirgGRP

GRP, the mammalian equivalent of bombesin (1,17), is a neurohormone with a plethora of effects in vivo and in vitro (28). GRP can act as a mitogen for some cell lines expressing its receptors (19, 26, 31). An autocrine loop involving the production of GRP and its receptor has been proposed to contribute to the transformation of small cell lung carcinomas (5). Various signal transduction mechanisms have been suggested for the bombesin/GRP receptor involving interactions between the receptor and a Gprotein (6,15) leading to activation of phospholipase C and phosphoinositide breakdown (8, 10, 30) and/or stimulation of transient protein tyrosine kinase activity (2,7). Using covalent cross-linking of radiolabeled ligand, the bombesin/GRP receptor has been putatively identified as a glycoprotein whose size is similar under reducing and nonreducing conditions, but varies depending on the cell line used for the analysis. The size of the bombesin/GRP receptor from mouse 3T3 cells ranges from 65 to 85 kDa (13,32), the size from the human glioma cell line GM 340 is 75 kDa (13) and from the rat pituitary cell line GH4C 1 is 100 kDa (29). The difference in size of the receptors from the mouse

~Present address: Department of Biomedical Sciences and Oncology, University of Torino Medical School, Corso Massimo D'Azeglio 52 10126 Torino, Italy. 2D.C. and L.N. are "Dottorandi di Ricerca" in Cytological and Morphogenetic Sciences from The University of Torino. 3present address: Department of Pharmacology, New York University Medical Center, 550 First Avenue, New York, NY 10016. '*Requests for reprints should be addressed to R. Kris at his present address: Department of Pharmacology, New York University Medical Center, 550 First Avenue, New York, NY 10016.

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CIRILLO ET AL.

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ABBREVIATIONS ATP BN BS3 CHAPS CHS DMEM DSS DST EDTA EGF EGS GDP GRP GTP GTPyS HEPES PAGE PBS PMSF SDS Sulfo-EGS

adenosine 5'-triphosphate bombesin bis (sulfosuccinimidyl) suberate 3-[(3-cholamidopropyl)dimethylammonio]- 1-propanesulfonic acid cholesteryl hemisuccinate Dulbecco's Modified Eagle's medium disuccinimidyl suberate disuccinimidyl tartarate ethylene diamine tetraacetic acid epidermal growth factor ethyleneglycolbis (succinimidylsuccinate) guanosine 5' diphosphate gastrin releasing peptide guanosine 5' triphosphate guanosine 5'-0-(3 thiotriphosphate) 4-(hydroxyethyl)-piperazine-ethanesulfonic acid polyacrylamide gel electrophoresis phosphate buffered saline phenylmethyl sulfonyl fluoride sodium dodecyl sulfate ethyleneglycolbis (sulfosuccinimidylsuccinate)

receptor is a complex of apparent molecular weight 220,000 containing the 75 kDa protein previously observed after crosslinking with [125I]GRP and using denaturing conditions. METHOD

Materials [Tyr4]Bombesin and GRP were purchased from Peninsula Laboratories. [125I]GRP was purchased from Amersham Corp. Other reagents were from the following sources: CHAPS (Calbiochem); CHS, PMSF, leupeptin, pepstatin, aprotinin, bacitracin and SDS markers (Sigma); EGS, DSS, DST, sulfo-EGS, BS 3 (Pierce). Water soluble molecular sieving chromatography markers were from Biorad. All the chemicals and solvents used were analytical or HPLC grade. Tissue culture products were purchased from Gibco.

Cells Swiss 3T3 and NIH 3T3 transfected fibroblasts were grown in Dulbecco's Modified Eagle medium supplemented with 10% (v/v) calf serum.

Preparation of Membrane Fraction From Swiss 3T3 Cells Confluent monolayers of fibroblasts were rinsed three times with ice-cold phosphate buffered saline (PBS) and scraped on ice with a rubber policeman in PBS containing 1 mM EDTA and 1 mM PMSF. Cells were then pelleted, washed once with a large volume of ice-cold lysis buffer with protease inhibitors (50 mM TRIS-HC1 p H = 8 . 0 , 1 mM EGTA, 5 mM MgC12, 50 p~g/ml leupeptin, 5 p,g/ml pepstatin, 10 pxg/ml aprotinin, 200 ~tg/ml bacitracin and 1 mM PMSF) and homogenized on ice in a Dounce homogenizer in the same buffer. For the removal of nuclei and other cellular debris, the homogenate was subjected to low-speed centrifugation (800 x g x 5' at 4°C), the supernatant was kept on ice while the pellet was reextracted with the same buffer and recentrifuged. The two supernatants were then pooled and centrifuged at 20,000 x g at 4°C for 30'. The crude membrane pellet was resuspended in HEMI buffer (25 mM HEPES-KOH, pH = 6.8, 5

mM MgCl2, l mM EGTA and protease inhibitors at the same concentration used in the lysis buffer). Protein concentration was measured with the protein assay reagent, BCA (Pierce). For storage at - 70°C, glycerol was added at a final concentration of 30% (v/v).

Cross-Linking on Swiss 3T3 Membranes Five hundred p~g of a Swiss 3T3 crude membrane preparation were incubated for 30 minutes at 37°C in HEMI buffer with 1.5 nM [125I]GRP in the presence or absence of 1 ~M unlabeled GRP. After the incubation, membranes were pelleted for 15 minutes at 1 8 , 0 0 0 x g at 4°C, washed once with ice-cold HEMI buffer, resuspended in ice-cold phosphate buffer, pH 7.0, containing 5 mM EGS (or 10 mM Sulfo-EGS, or 10 mM BS3, or 1 mM DSS, or 1 mM DST) and incubated for 20 minutes on ice. The reaction was stopped by addition of TRIS, pH 7.6 (20 mM final concentration). Binding experiments on membranes were performed in HEMI buffer in the presence of 1 nM of [~25I-Tyr4]bombesin as previously described (21).

Solubilization of Swiss 3T3 Membranes Under Nondenaturing Conditions Membrane preparations either covalently labeled with [~25I]GRP or untreated were solubilized for 1 hr at 4°C at a protein concentration of 15 mg/ml with 0.75% CHAPS (w/v) and 0.15% cholesteryl hemisuccinate (CHS) (w/v) in HEMI buffer in the presence of 10% (v/v) glycerol to improve the stability of solubilized receptor (21). Extracts were cleared by ultracentrifugation (150,000 × g) for 30 minutes at 4°C and diafiltrated in a Centricon 30 (Amicon) for several cycles to remove the free ligand and lower the detergent concentration to 0.1%.

Sucrose Densi~ Gradient Sedimentation Linear sucrose density gradients (5-25%) were prepared in HEMI buffer containing 0.1% CHAPS with or without 0.02% CHS. Solubilized [125I]GRP-labeled membranes (100 ~1) were applied on a 5-ml gradient• The gradients were run in a Beckman SW 50.1 rotor for 15 hr at 35,000 rpm at 4°C. Fractions containing 3 drops were collected from the bottom and counted in an LKB gamma counter (60% counting efficiency). Fractions containing the peaks were concentrated in Centricon 30, and analyzed by SDS PAGE after addition of Laemmli buffer (14).

Molecular Sieving Chromatography The detergent-soluble fraction (0.2 mg of solubilized proteins) • • 125 from Swiss 3T3 membranes covalently labeled with [ I]GRP were loaded on a TSK 4000 preparative column, The column was preequilibrated with 20 mM HEPES, p H = 6 . 8 , with 5 mM MgC1 z, 10% (v/v) glycerol (HMG) and different concentrations of CHAPS plus one fifth its concentration of CHS and run at a flow rate of 3 ml/min. One-minute fractions were collected and counted in an LKB gamma counter. One-ml aliquots of the radioactivity containing fractions were concentrated and analyzed by SDS gel electrophoresis to detect the [~25I]GRP covalently labeled species. The column was calibrated under identical conditions using Biorad water-soluble markers and labeled EGF receptor (170 kDa) and an EGF receptor truncated mutant (80 kDa). NIH 3T3 fibroblasts stably transfected with EGF receptor and the truncated mutant were solubilized under the same conditions used for the analysis of the GRP receptor from Swiss 3T3 fibroblasts and the extract loaded on the HPLC column. The fractions were then

DETERGENT SOLUBILIZED GRP RECEPTOR

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Fraction N u m b e r FIG. 1. Molecular sieving chromatography of CHAPS extract of [12SI]GRP covalently labeled Swiss 3T3 membranes. [125I]GRP covalently labeled Swiss 3T3 membranes were analyzed by molecular sieving chromatography as a TSK preparative column after solubilization under nondenaturing conditions (see the Method section). Void volume (Vo) is the elution volume of blue dextran. Arrows show the elution volume of the EGF receptor (170 kDa) and truncated EGF receptor mutant (80 kDa) used as markers. The inserts show an SDS PAGE analysis of the peaks. The number of the original fraction of the chromatography profile is indicated.

immunoprecipitated with anti-EGF receptor antibodies (RK2) (12) and phosphorylated with [32P]~,ATP. The kinase reaction was stopped by the addition of boiling Laemmli buffer. SDS PAGE analysis was performed and the gels dried and autoradiographed at - 7 0 ° C with Kodak-X-Omat film and enhancing screens.

Soluble Receptor Assay Swiss 3T3 untreated membranes extracted at 10 mg/ml were loaded on a TSK 4000 preparative column and run in HMG buffer with 0.1% CHAPS and 0.02% CHS at 3 ml/minute flow rate. One-minute fractions were collected. Collected fractions (500 txl) were assayed for the presence of GRP receptor by incubation with 1 nM [~25I-Tyr4]bombesin for 3 hr on ice. Protein bound radioactivity was separated from free ligand by vacuum suction through Whatman GF/F glass fiber filters pretreated with 0.3% polyethyleneimine (v/v) (Eastman Kodak) in H20 for 2 hours. Filters were washed twice with 8 ml of ice-cold 20 mM HEPES, pH = 6.8, 5 mM MgC12 and then counted with an LKB gamma counter (21). For each fraction, the assay was conducted in triplicate. Specific binding was calculated by subtracting nonspecific binding, which was the amount of [125I-Tyr4]bombesin bound in the presence of 1 txM unlabeled [Tyr4]bombesin from the total binding.

Inhibition of [1251-Tyr4]Bombesin Binding and [lzsI]GRP CrossLinking by Nucleotides Swiss 3T3 crude membrane preparation (150 Ixg) was sus-

pended in a final volume of 100 i.tl of HEMI buffer and incubated with 0.5 nM [125I-Tyr4]bombesin for 30 minutes at 37°C in the presence or in the absence of different concentrations of nucleotides. At the end of the incubation, samples were quickly filtered under vacuum suction through Whatman GF/C glass fiber filters, pretreated with 0.3% polyethyleneimine in H20 for 2 hours. Filters were washed twice with 8 ml of ice-cold 20 mM HEPES, pH 6.8, and 5 mM MgC12 and counted in an LKB gamma counter. Specific binding was calculated by subtracting the nonspecific (determined as binding in presence of 1 ixM unlabeled [Tyr4]bombesin) from total bound. Inhibition of both [12SI]GRP binding and cross-linking by nucleotides was performed by incubating the same amount of Swiss 3T3 membranes with 0.5 nM [t25I]GRP in HEMI buffer in the presence of 10 ~M GTP~/S, 100 IxM GTP, or 100 p.M ATP, for 30 minutes at 37°C. Membranes were washed twice with ice-cold HEMI buffer in an Eppendorf centrifuge, resuspended in phosphate buffer, pH 7.0, and crosslinked with 5 mM EGS for 30 minutes on ice. The reaction was stopped by adding concentrated Laemmli buffer. Samples were electrophoresed in SDS PAGE and the gel dried and autoradiographed at - 7 0 ° C with Kodak-X-Omat films and enhancing screens.

Protein Tyrosine Kinase Assay on Fractions From Molecular Sieving Chromatography Quiescent Swiss 3T3 cells were stimulated with 5 nM bombesin in the spent medium for 3 minutes at 37°C. The medium

CIRILLO ET AL.

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FIG. 2. Rechromatography of the pooled fractions from peak 1 and peak 2. (a) Fractions containing peak 1 were pooled and rechromatographed with no treatment ( , ) or after treatment with 1% boiling SDS (0). (b) Rechromatography of pooled fractions from peak 2: (m) no treatment or (O) treatment with 1% boiling SDS.

solubilized with the zwitterionic detergent CHAPS and analyzed by HPLC molecular sieving chromatography on a TSK 4000 column. The protein bound radioactivity eluted in two peaks: a more prominent peak 1 of approximately 220 kDa and a smaller peak 2 of approximately 80 kDa. The column was calibrated with CHAPS solubilized [32p]-labeled EGF receptor (170 kDa) and a truncated EGF receptor mutant (80 kDa) (Fig. 1). Concentrated material from peak fractions was analyzed using SDS PAGE. The 75 kDa protein was the major covalently labeled species in both peaks (Fig. 1, insert). When the binding and cross-linking of [~25I]GRP to its receptor was performed in the presence of different concentrations of unlabeled GRP there was a dosedependent decrease in the amount of radioactivity in the peaks (data not shown). Several bifunctional cross-linking agents were tested on Swiss 3T3 membranes. The elution profile after solubilization in detergent was not affected by the molecular length or the solubility properties of the cross-linker used. To determine if there was an equilibrium between peaks 1 and 2, pooled fractions from each peak were rechromatographed. Radioactivity from each peak eluted with the original volume, showing no apparent movement of one form to another under these conditions (Fig. 2). Treatment of peak 1 fractions with 1% boiling SDS before rechromatography resulted in the disappearance of peak 1 and the movement of the radioactivity to an elution volume corresponding to peak 2 plus a novel peak of approximately 30 kDa (peak 3). After SDS treatment of peak 1, the 75 kDa cross-linked receptor was only evident in the peak corresponding to peak 2 and was not present in peak 3. Rechromatography of peak 2 after SDS treatment showed no change (Fig. 2). The nature of the 30 kDa component is presently unknown. Protease inhibitors were added in the solubilization procedure and were further added to the tubes for fraction collection to rule out degradation. Moreover, rechromatography of peak 1 did not give rise to the 30 kDa species, suggesting degradation of peak 1 does not produce the 30 kDa species.

Sucrose Density Gradient Sedimentation of Solubilized Swiss 3T3 Membranes Covalently Labeled With [1251]GRP was removed, the monolayers were quickly rinsed in ice-cold HEMI buffer and extracted in 0.75% CHAPS, 0.125% CHS in HEMI buffer with tyrosine phosphatase inhibitors (100 txM NaVO3, 100 mM Na F) for 30 minutes on ice. The soluble material was cleared at 150,000 × g for 30 minutes at 4°C, diluted to 0.1% detergent with HEMI buffer and chromatographed under the same conditions used for the []25I]GRP cross-linked solubilized membranes. Fractions were immunoprecipitated with antiphosphotyrosine antibodies (3)-protein A-sepharose complexes and the kinase assay was performed by exposing the immunoprecipitates to [32P]~/ATP for 5 minutes at 30°C in the presence of 10 mM MnC12. The reaction was stopped with boiling Laemmli buffer. Samples were subjected to SDS PAGE to separate the [32p]-labeled proteins, and the gels were dried and autoradiographed with Kodak-X-Omat films and enhancing screens. RESULTS

Molecular Sieving Chromatography of Solubilized Swiss 3T3 Membranes Covalently Labeled With [1251]GRP []25I]GRP was covalently cross-linked after equilibrium binding to a crude Swiss 3T3 membrane preparation, using the homobifunctional cross-linker EGS. SDS PAGE analysis of an aliquot showed the prominent labeling of a 75 kDa protein, as previously described (32). The radiolabeled material was then

Sucrose density gradient sedimentation analysis of solubilized Swiss 3T3 membranes covalently labeled with []25I]GRP showed 2 peaks of protein-associated radioactivity (Fig. 3). SDS PAGE analysis of the fractions containing the two peaks showed the previously described 75 kDa GRP binding moiety as the major labeled species (Fig. 3, insert). Gradients were standardized using [32p]-labeled EGF receptor and a truncated EGF receptor mutant. Running the gradient in a higher concentration of detergent or omitting the cholesteryl compound from the buffer did not affect the results (data not shown). The use of DSS or the shorter DST instead of EGS as cross-linking agents had no effect on the sedimentation pattern (data not shown).

Molecular Sieving Chromatography of Functional Receptors Solubilized From Swiss 3T3 Membranes Swiss 3T3 membranes were preincubated to equilibrium with []25I-Tyra]bombesin and solubilized in CHAPS under nondenaturing conditions. The extract was subjected to molecular sieving chromatography under the same conditions used for the crosslinked receptor. A peak of radioactivity eluted at the same position as peak 1 using cross-linked receptor (Fig. 4a). This peak was specifically inhibited when the membranes were preincubated with a thousand-fold excess of unlabeled ligand. Most of the nonspecific binding eluted as large aggregates close to the void volume

DETERGENT SOLUBILIZED GRP RECEPTOR

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(data not shown). [~2SI]GRP also bound specifically to the same fraction (data not shown). In another set of experiments, the fraction collected from molecular sieving chromatography of a membrane extract was assayed for the presence of soluble GRP receptor. Specific [~25I-Tyra]bombesin binding activity eluted with a volume corresponding to peak 1 (Fig. 4b). The use of [~25I]GRP in similar experiments gave the same results but with higher background binding as expected for the more hydrophobic GRP (data not shown).

Effects of Guanine Nucleotides A dose-dependent inhibition of up to 50% of the specific binding of [125I-Tyr4]bombesin to Swiss 3T3 membranes was exerted by guanine nucleotides. The nonhydrolyzable analogue GTY~/S was the most potent inhibitor with a maximum inhibition of 50% at 10 I~M, GTP was less effective with 30% inhibition at 100 IxM and GDP caused a slight decrease from the level of control, untreated samples. Samples treated with ATP at the same concentration had no effect (Fig. 5). When cross-linking of [~25I]GRP on Swiss 3T3 membranes was performed in the presence of guanine nucleotides at concentrations maximally effective for the inhibition of ligand binding, SDS PAGE analysis showed a marked inhibition of the labeling of the 75 kDa protein. Effectiveness and potency of the nucleotides tested paralleled those observed for the inhibition of ligand binding (Fig. 6). Molecular sieving chromatography of detergent solubilized mem-

branes, covalently labeled with [~25I]GRP in the presence of GTP~/S, showed no differences in the elution volume of both peak 1 and peak 2.

Assay of Bombesin-Dependent Protein-Tyrosine Kinase Activity in Fractions From Molecular Sieving Chromatography of Swiss 3T3 Membrane Extract It was previously shown that bombesin stimulation of Swiss 3T3 cells leads to activation of a protein-tyrosine kinase activity whose major phosphorylation product is a protein of 115 kDa (p115). To access whether the protein kinase activity responsible for p115 phosphorylation was associated in a complex with GRP receptor, confluent cultures of Swiss 3T3 cells were stimulated with a fully mitogenic dose of bombesin and extracted with CHAPS using a mixture of phosphotyrosine phosphatase inhibitors. The cleared extract was then resolved by molecular sieving chromatography on a TSK 4000 column as described for the cross-linked receptor, with the addition of the phosphotyrosine phosphatase inhibitors in the elution buffer. Fractions were collected and analyzed for the presence of bombesin-dependent protein-tyrosine kinase activity. A prominent labeling of the 115 kDa protein was observed in fractions eluting between the void volume and the position of peak 1 in the chromatograph (Fig. 7). DISCUSSION

Interaction between GRP and its receptor triggers a cascade of

742

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FIG. 4. Molecular sieving chromatography of functional receptors solubilized from Swiss 3T3 membranes. (a) Swiss 3T3 membranes were solubilized under nondenaturing conditions after equilibrium labeling with [125I-Tyr4]bombesin and subjected to molecular sieving chromatography. Total binding is shown. (b) Swiss 3T3 membranes were solubilized under nondenaturing conditions and subjected to molecular sieving chromatography. Fractions were collected and analyzed for [125I-Tyr4]bombesin binding activity as described under the Method section. Specific binding is shown. Specific binding was calculated by subtraction of binding in the presence of 1 I~M unlabeled [Tyrn]bombesin from the total binding.

events leading to a variety of cellular responses depending on the cell type expressing the receptor. In mouse 3T3 fibroblasts, the interaction between bombesin or GRP and its receptor induces changes in membrane ionic fluxes (18), increase in intracellular Ca ++ (9, 20, 30), cytoplasm alkalinization (18), phosphoinositide turnover (10,30), serine and tyrosine phosphorylation of cellular proteins (2,11 ), transcription of cellular oncogenes (15,23), and eventually cell division. One proposed mechanism for the transfer of the growth signal from GRP receptor to effector

molecules is via interaction with a G-protein. The evidence for a G-protein involvement includes the inhibition of the binding of radiolabeled ligand to membrane preparation from Swiss 3T3 cells by guanine nucleotides [(6,15) and Fig. 5]. While bombesininduced mitogenesis is inhibited by pertussis toxin (15), no effect of the toxin on the modulation of ligand binding by guanine nucleotides was observed (6). It remains to be shown whether a G-protein is responsible for the coupling of GRP receptor to phospholipase C, which in turn affects phosphoinositide turnover. A specific bombesin-stimulated protein-tyrosine kinase activity has also been detected in Swiss 3T3 cells and small cell lung carcinoma cells lines (2,7), leading to phosphorylation on tyrosine of a 115 kDa protein. However, another group (11) was unable to detect a marked increase in tyrosine phosphorylation after bombesin stimulation. To obtain structural information on GRP receptor, membrane preparations from Swiss 3T3 cells were labeled with [J25I]GRP and bifunctional cross-linkers. SDS PAGE analysis showed a major 75 kDa protein specifically labeled as previously described. Its labeling was inhibited by guanine nucleotides in a similar fashion to that observed for the inhibition of binding of radiolabeled ligand to the receptor, with the nonhydrolyzable GTP analogue GTP~S more potent than GTP. Upon solubilization with zwitterionic detergent using conditions previously shown to preserve the binding properties of the receptor, the 75 kDa [125I]GRPlabeled protein eluted in the same volume as the specific binding activity of GRP receptor using molecular sieving chromatography. These results give further evidence to the proposed role of the 75 kDa protein as the binding moiety of GRP receptor. The receptor solubilized under these conditions is comparable to the membrane bound receptor with respect to ligand affinity and pharmacological binding profiles (21). However, the sedimentation profile in a sucrose density gradient and the elution volume in molecular sieving chromatography

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either by the residual bound radiolabeled ligand or by soluble receptor assay on the collected fractions eluted in the same volume as the larger peak (peak 1). Correct determination of the molecular weight of GRP receptor must take into account the association of the nonionic detergent to the solubilized proteins. We addressed this issue by calibration of the gel permeation column with labeled membrane proteins solubilized under the same condition. A similar density of the protein detergent complex was assumed for both markers and cross-linked receptor as supported also by the preservation of their relative positions in sucrose density sedimentation. Given the limits of such assumptions, apparent molecular weights of 220,000 and 80,000 were estimated for the two peaks. Other studies have similarly shown larger sizes for membrane receptors solubilized with nonionic detergents and analyzed by gel filtration chromatography than expected from the data collected from cross-linking with radiolabeled ligand and SDS PAGE analysis (4, 22, 25). In the case of VIP receptor solubilized from lung membranes, a larger size molecular complex containing high affinity receptors was reported after CHAPS solubilization compared to the 55 kDa determined by SDS PAGE analysis of the [1zsI]VIP cross-linked receptor (25). This high affinity receptor complex was shown to be sensitive to GTP and its analogue Gpp(NH)p induced inhibition of binding, suggesting the participation of a G-protein to this complex (25). A relatively stable association of G-proteins and receptors was also observed on solubilized liver VIP receptor [3-adrenergic and adenosine receptor (16,27). Alternatively, the large molecular weight estimated for the solubilized VIP receptor using water-soluble markers as standards can be entirely due to the amount of detergent bound to such a hydrophobic protein (24). However, the partition of the cross-linked 75 kDa GRP receptor between a high and low molecular weight form, with the lower one being close to the size calculated from SDS PAGE analysis, argues against such an interpretation. In fact, it is unlikely that the same protein is able to interact in two different ways with detergent. It remains to be seen whether the low molecular weight form represents a physiological state of the receptor or may instead be a product of the solubilization protocol devoid of binding activity, as indicated by the soluble receptor assay. In addition, no major change in the elution profile was observed varying the concentra-

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