©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Leydig Cell cDNA-derived Protein Is a Relaxin-like Factor (*)

Erika E. Büllesbach , Christian Schwabe

From the (1)Department of Biochemistry and Molecular Biology Medical University of South Carolina, Charleston, South Carolina 29425

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

According to Burkhardt et al. (Burkhardt, E., Adham, I. M., Brosig, B., Gastmann, A., Mattei, M. G., and Engel, W.(1994) Genomics 20, 13-19) Leydig cells contain the message for a protein of the insulin/relaxin family which was named Leydig cell insulin-like protein (LEY I-L). We have synthesized the human LEY I-L according to the amino acid sequence deduced from the published cDNA structure and obtained preliminary results concerning its potential target organs and its biological activity. Leydig cell insulin-like protein binds specifically to crude membrane preparations of mouse uterus and brain and shows cross-reactivity with the relaxin receptor, but not the insulin receptor. On the basis of these observations, together with the results of earlier structure-function considerations, we suggest that the new protein is a relaxin-like factor. By itself the new factor shows no obvious effect, but when given together with relaxin it significantly enhances relaxin-mediated widening of the mouse symphysis pubis.


INTRODUCTION

Relaxin, the Leydig cell insulin-like protein(1, 2) , and insulin share general structural features such as molecular weight, the two-chain structure, and the number and disposition of disulfide links. Large sequence differences exists between relaxins from different species (3) and between relaxins, the new factor, and insulins(1) . The fine structural differences of these two chain proteins, however, depend critically upon only a few type-specific amino acid residues such as the glycine in position A14 (human relaxin II and the relaxin-like factor) or the isoleucine in the equivalent position (A10) in insulin(3) . The receptor-binding site of relaxin is comprised of a cassette of the general structure Arg-X-X-X-Arg which is located in the midsection of the B chain helix in positions B13 to B17 in human relaxin II and in the same relative position in all relaxins (4). This cassette is also present in the new protein but offset toward the C terminus by precisely four residues. In these critical regions the new factor resembles relaxin rather than insulin. Here we are reporting the synthesis of the new factor and the experiments that support our contention that this factor be called relaxin-like factor (RLF)()rather than insulin-like protein.


EXPERIMENTAL PROCEDURES

Materials

L-Amino acid derivatives for peptide synthesis were purchased either from Bachem Bioscience (Philadelphia, PA) or Bachem California (Torrance, CA). Solvents for peptide synthesis and chromatography were distilled in high purity solvent (Burdick and Jackson; Muscagon, MI), and the chemicals for peptide synthesis were obtained from Perkin-Elmer Applied Biosystems (Foster City, CA). Other chemicals of analytical grade were used without further purification.

Methods

Peptide Synthesis

The B chain of Leydig cell-derived protein was synthesized by tert-butyloxycarbonyl chemistry using conventional HF-labile side chain-protecting groups for all three functional amino acids except cysteines. Cysteine B10 was protected by the acetamidomethyl group and B22 by the thiol-protecting/activating group [S-(3-nitro-2-pyridinesulfenyl)] (Cys). Methionine was protected by sulfoxide formation and tryptophan by the N(in)-formyl group. The synthesis was performed on an Applied Biosystems peptide synthesizer model 430A on 4-(oxymethyl)phenylacetamidomethyl resin loaded with 0.4 mmol of tert-butyloxycarbonyl-alanine. Deprotection and removal from the solid support was accomplished by HF treatment in the presence of 5% m-cresol. The crude peptide was extracted with 20% acetic acid and lyophilized (yield 1.387 g). The B chain was purified on Sephadex G50-sf (2.5 50 cm) in 1 M acetic acid (yield: 840 mg), followed by preparative HPLC on Synchropak RP-P (2.1 25 cm) in portions of 50-70 mg. The column was equilibrated in 20% solvent B and the peptide eluted with a linear gradient of 20% solvent B to 50% solvent B over 1 h at a flow rate of 5 ml/min (overall yield: 233 mg). Amino acid composition: Thr, 2.00(2) ; Ser, 0.86(1) ; Glu, 2.90(3) ; Pro, 3.09(3) ; Gly, 3.28(3) ; Ala, 2.16(2) ; Cys, 0.89(2) ; Val, 3.19(3) ; Met, 1.22(1) ; Leu, 1.94(2) ; Phe, 0.99 (1); His, 2.44(2) ; Lys, 0.96(1) ; Arg, 3.81(4) .

The A chain (0.25 mmol) was synthesized via Fast-moc chemistry on an ABI peptide synthesizer (model 430A) on p-benzyloxybenzyl resin. All side chains were protected by trifluoroacetic acid-labile protecting groups except Cys, which was Acm-protected, and Cys which was protected by the HF-labile p-methylbenzyl group. The A chain was deprotected with trifluoroacetic acid/thiophenol (10:1, v/v), using 50 mg of peptidyl resin/ml for 90 min at room temperature(5) . The trifluoroacetic acid was evaporated and the peptide precipitated with ether. The precipitate was collected by centrifugation, the supernatant discarded, and the pellet washed twice with ether and air-dried. The peptide was suspended in water, dissolved by the addition of ammonia, and desalted on Sephadex G-25m in 50 mM NHHCO. To the eluate (100 ml) 50 ml of MeSO was added in order to accelerate the oxidation of the intrachain disulfide bond A10-A15(6) . The progress of oxidation was observed by the Ellman reaction(7) . After completion of the disulfide bond formation the A chain was dialyzed against water and lyophilized (yield: 372.3 mg). Aliquots of 20 mg were further purified by preparative HPLC on Synchropak RP-P (10 250 mm). The column was equilibrated in 30% solvent B and the peptide eluted with a linear gradient of 30% solvent B to 50% solvent B over 30 min at a flow rate of 3 ml/min (overall yield: 166.5 mg). Amino acid composition: Asp, 2.20(2) ; Thr, 3.00(3) ; Ser, 0.99(1) ; Glu, 1.92(2) ; Pro, 2.25(2) ; Gly, 1.06(1) ; Ala, 4.18(4) ; Cys, 1.62(4) ; Leu, 3.60(4) ; Tyr, 1.82 (2); Arg, 0.98(1) .

For chain combination 33.4 mg (11.3 µmol) of the A chain (AcmA10,MBzlA24) was treated with 4 ml of HF in the presence of 200 µl of m-cresol for 45 min at 0 °C. Thereafter the HF was evaporated in a stream of nitrogen and the peptide precipitated with ether. The pellet was collected and dried over KOH in vacuo for 30 min. The monothiol A chain was dissolved in 4 ml of 8 M guanidinium chloride in 0.1 M acetic acid at pH 4.5 and added to 36.3 mg (9.6 µmol) of the B chain. The disulfide bond A24-B22 was formed at 37 °C for 24 h and the resulting product separated first on Sephadex G50-sf in 1 M acetic acid (column 2.5 50 cm) (yield: 48.7 mg, 78.3%), followed by preparative HPLC on Synchropak RP-P (10 250 mm). The column was equilibrated in 30% solvent B and the peptide eluted with a linear gradient of 30-45% solvent B over 30 min at a flow rate of 3 ml/min (yield: 34.1 mg, 54.8%).

The resulting peptide contained acetamidomethyl groups in positions Cys and Cys, the N(in)-formyl group in Trp, and a sulfoxide in the side chain of Met. For the formation of the third disulfide bond the peptide (9.3 mg) was dissolved in water (3.5 ml) and added to a stirred solution consisting of acetic acid (3.5 ml) 6 N HCl (19.1 µl) and 3 ml of 50 mM iodine in acetic acid(8) . The reaction was performed at room temperature for 10 min, quenched with ascorbic acid, and the product was desalted on Sephadex G25-sf in 1 M acetic acid and lyophilized. After purification by preparative HPLC (conditions as before) (yield: 3.42 mg, 36.8%) the protein still contained protecting groups in Trp (B27) and Met (B5).

Complete deprotection was achieved first by treatment of 11.3 mg of the peptide with 2 ml of water/piperidine 9:1 (v/v) for 2 min at room temperature. The base was neutralized with 0.4 ml of acetic acid and the peptide purified by preparative HPLC, dried (yield: 11.0 mg, 97.5%), and 10 mg of peptide-containing methionine sulfoxide was reduced with 1 ml of trifluoroacetic acid, 0.5 M NHI in water 9:1 (v/v) for 15 min at 0 °C. Free iodine was reduced with 0.5 M ascorbic acid in water and the reaction quenched by dilution with water. The final peptide was recovered by preparative HPLC (conditions as before) (yield: 7.57 mg, 75.7%). Amino acid composition: Asp, 2.02(2) ; Thr, 4.79(5) ; Ser, 1.77(2) ; Glu, 4.86(5) ; Pro, 5.17(5) ; Gly, 4.15(4) ; Ala, 6.09(6) ; Cys, 3.51(6) ; Val, 2.86(3) ; Met, 0.70(1) ; Ile, 0(0) ; Leu, 5.74(6) ; Tyr, 2.12(2) ; Phe, 0.98(1) ; His, 2.00(2) ; Lys, 1.26(1) ; Trp, 1.00 (1); Arg, 5.02 (5) (overall yield 14.9%).

HPLC

The mobile phase of all HPLC systems used consisted of 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in 80% acetonitrile (solvent B).

For preparative HPLC a Waters HPLC system consisting of two pumps (model 6000A) and gradient programmer (model 680) was used in combination with a Synchropak RP-P column (C18) (SynChrom, Inc.) and an Uvicord S UV (226 nm) monitor (LKB, Bromma, Sweden). Usually 1-20 mg of peptide was separated using linear gradients as indicated.

Analytical HPLC I

Analytical HPLC I was performed on Aquapore 300 (C; 2.1 30 mm) using an Applied Biosystems HPLC model 130A. Separation was achieved with a linear gradient from 23 to 34% solvent B in 60 min at a flow rate of 0.1 ml/min. The peptide was detected by UV absorbance at 230 nm.

Analytical HPLC II

Analytical HPLC II was performed on Synchropak RP-P (C18, 4.1 250 mm) using a Waters HPLC system. Separation was achieved with a linear gradient from 20% solvent B to 50% solvent B in 30 min at a flow rate of 1 ml/min. The peptide was detected by UV absorbance at 220 nm.

Amino Acid Analyses

Peptides were hydrolyzed in vapor phase 6 N HCl containing 0.1% phenol for 1 h at 150 °C. The amino acids were detected after pre-column modification with phenylisothiocyanate and separation by HPLC (PicoTag system, Waters Millipore).

Sequence Analyses

Sequence analyses were performed on an ABI 477A pulsed liquid protein sequencer and an in-line ABI 120A phenylthiohydantoin analyzer (ABI, Applied Biosystems, Foster City CA). Chains were prepared by reduction of about 10 µg of the relaxin-like factor in 20 µl of 50 mM dithiothreitol in 3 M guanidinium chloride, 0.2 M Tris/HCl at pH 8.5 for 1 h at 37 °C, diluted with 30 µl of solvent A, followed by separation on Aquapore 300 (see ``Analytical HPLC I'' and ``Analytical HPLC II'' for conditions).

UV Spectroscopy

UV spectroscopy was performed on an OLIS Cary 15 spectrophotometer conversion (On-Line Instrument Systems Inc., Bogart, GA). UV spectroscopy was used to determine the protein concentrations of Leydig cell-derived protein. The specific absorption coefficient ( = 1.40 cm/mg) was obtained by direct comparison of UV absorbance and the recovery of amino acids after hydrolysis and amino acid analysis.

Circular Dichroism

CD spectroscopy was performed on a Jasco J-710 spectropolarimeter using a cell of 0.02-cm path length. Proteins were dissolved in 25 mM Tris/HCl at pH 7.7, and concentrations were determined by UV spectroscopy: 0.67 mg/ml for porcine relaxin, 0.54 mg/ml for relaxin-like factor, and 0.55 mg/ml for human relaxin. Spectra were measured at a resolution of 0.2 nm, a bandwidth of 2 nm, and five spectra were averaged. Molar ellipticity was calculated according to Adler et al.(9) using mean residual weights of 110.4 for relaxin-like factor, 113.6 for porcine relaxin, and 112.5 for human relaxin.

Mass Spectrometry

Mass spectra were recorded on a Jeol HX110/HX110 4 sector tandem mass spectrometer (Jeol, Tokyo, Japan). Samples were dissolved in 0.1% trifluoroacetic acid at a concentration of about 0.8 nmol/µl.

I-Labeled Relaxin-like Factor

For tracer preparation a synthetic precursor of the relaxin-like factor was used which contained side chain-protected tryptophan and methionine. The peptide, 10 µg in 5 µl of water, was placed into a 200-µl Eppendorf vial, 5 µl of phosphate buffer (250 mM, pH 7.4) was added, followed by 2 µl of I (1 mCi), and 5 µl of chloramine T (2 mg/ml in phosphate buffer, pH 7.4). The reaction was performed for 1 min on ice, quenched by addition of 5 µl of sodium thiosulfate (5 HO) (50 mg/ml in phosphate buffer, pH 7.4), and 5 µl of NaI (20 mg/ml in phosphate buffer, pH 7.4). The side chain-protecting group of Trp was removed by addition of 5 µl of piperidine. After 2 min at room temperature the reaction was quenched by the addition of 5 µl of glacial acetic acid, the reaction mixture was diluted with 10 µl of water and loaded onto a Aquapore 300 column for separation. The protein was detected by UV absorbance, and peaks were manually collected into 100 µl of 1% bovine serum albumin in water.

Receptor Binding Assays

Insulin-receptor binding assays were performed on crude membrane preparations of term placenta (10) using [I]iodo-Tyr-porcine insulin as tracer(11) . Assays were performed in HMS buffer (25 mM HEPES, 104 mM NaCl, 5 mM MgCl, 0.2% bovine serum albumin, pH 7.4) in a total volume of 100 µl. Labeled insulin (50,000 cpm/assay, 150 pM), and variable amounts of insulin were incubated with crude membranes for 1 h at room temperature. Thereafter 1 ml of buffer was added, the membranes collected by centrifugation in a microcentrifuge at 14,000 rpm for 5 min, the supernatant discarded, and the tip of the Eppendorf vial cut off and counted in a -counter (Minigamma, LKB, Bromma, Sweden). To determine nonspecific binding, unlabeled insulin was used at a concentration of 2 µg/ml (0.33 µM), and nonspecific binding was usually below 10% of the total binding.

Relaxin Binding Assays

Relaxin binding assays were performed as described (12, 13) using crude membrane preparations of mouse tissue. Mouse brains of two mice were collected into 15 ml of chilled buffer (25 mM HEPES, 0.14 M NaCl, 5.7 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, and 80 mg/ml soybean trypsin inhibitor, pH 7.5) supplemented with sucrose (0.25 M, final concentration). The tissue was homogenized on ice for 10 s with a Polytron homogenizer (Brinkmann Instruments) at setting 5. The homogenate was centrifuged at 700 rpm for 10 min at 4 °C, and the supernatants were recentrifuged at 10,000 g for 1 h. The pellet was resuspended in 15 ml of ice-cold binding buffer, 25 mM HEPES, 0.14 M NaCl, 5.7 mM KCl, 0.2 mM phenylmethylsulfonyl fluoride, and 80 mg/ml soybean trypsin inhibitor, pH 7.5, supplemented with 1% bovine serum albumin, and centrifuged for 1 h at 10,000 g. The crude membrane preparation was suspended in 1 ml of binding buffer and 40 µl was used per assay. The assay was performed using 40 µl of tracer (about 100,000 cpm of porcine relaxin tracer = 150 pM) and 20 µl of relaxin at various concentrations. The assay was incubated for 1 h at room temperature, and the suspension diluted with 1 ml of wash buffer (25 mM HEPES, 0.14 M NaCl, 5.7 mM KCl, 1% bovine serum albumin, 0.01% NaN) and centrifuged in an Eppendorf centrifuge at 14,000 rpm for 10 min. The supernatant was discarded and the tip of the vial cut and counted in a -counter. Nonspecific binding was determined in the presence of 2 µg/ml of unlabeled competitor (0.33 µM). In a typical experiment the specific binding was between 25 and 40% of the total binding.

The relaxin-like factor was assayed either on crude membrane preparations of human placenta under the conditions described for insulin or on crude membrane preparations of mouse tissues using the assay system described for relaxin. Tissue specificity was determined using crude membrane preparations of leg muscles, kidneys, liver, brain, and uterus (of estrogen-primed mice). The crude membranes were prepared as described for relaxin. Binding was based on protein concentration determined by Lowry.

Mouse Symphysis Pubis Assay

Mouse interpubic ligament assays were performed essentially as described by Steinetz et al.(14) . Ovariectomized virgin female mice were primed with 5 µg of estrogen cypionate in 100 µl of sesame oil. Five days later the mice were injected subcutaneously with human relaxin(15) , RLF, or mixtures of human relaxin and RLF in 100 µl of 0.1% benzopurpurin 4B. For negative control 100 µl of 0.1% benzopurpurin 4B in water were injected. After 16 h the mice were killed in an atmosphere of CO the symphysis pubis dissected free, and the distance between the interpubic bones measured with a dissecting microscope fitted with transilluminating fiber optics.


RESULTS AND DISCUSSION

The synthesis of the human Leydig cell-derived protein (Fig. 1) has been achieved by solid phase methodologies in combination with site-directed sequential disulfide bond formation (Fig. 2). Correctness of the disulfide bonding pattern is an intrinsic result of the cross-linking technique and has been confirmed in many previous syntheses of relaxins and insulins(4, 13, 15, 16, 17, 18) .


Figure 1: The primary structure of the relaxin-like factor is compared with the sequences of human relaxin and insulin. Note the relative positions of the B chain arginines (highlighted) in RLF versus relaxin. Insulin does not contain these residues. In the A chain loop of RLF and relaxin the relaxin-specific glycine is seen. The corresponding residue in insulin is isoleucine.




Figure 2: Schematic of the site-directed sequential disulfide link formation. 1) Trifluoroacetic acid deprotection; 2) oxidation of the thiols using MeSO/50 mM NHHCO (1:2, v/v); 3) HF deprotection of Cys(MBzl); 4) combination of A and B chains at pH 4.5 in 8 M guanidinium chloride; 5) formation of the third disulfide link by reaction with iodine in 70% acetic acid; 6) liberation of the tryptophan side chain with 10% piperidine; 7) reduction of methionine sulfoxide with 33-fold excess of NHI in 90% trifluoroacetic acid. CHO, N(in)formyl; Npys, [S-(3-nitro-2-pyridinesulfenyl)]; O, sulfoxide.



The purity of the RLF was verified by reverse phase high performance liquid chromatography (Fig. 3) and amino acid analysis. Upon reduction two chains were generated, isolated, and sequenced; both chains had the correct primary structure. Partial hydrolysis at the acid-labile Asn-Pro bond was not detected, and mass spectrometry showed the correct ion for the synthetic RLF (found: m/z 6294.6; theoretical: m/z 6293.2). Comparative circular dichroic spectra suggested near identity of the solution structures of RLF and porcine relaxin (Fig. 4). Human relaxin (Fig. 4) and insulin (data not shown) have an absolute minimum at a slightly longer wavelength and, in case of insulin, of lesser intensity(19) .


Figure 3: HPLC record of the purified RLF. Chromatography was performed on Synchropak RP-P (4.1 250 mm) using a linear gradient from 20 to 50% solvent B in 30 min (solvent A: 0.1% trifluoroacetic acid in HO and solvent B: 0.1% trifluoroacetic acid in 80% acetonitrile) at a flow rate of 1 ml/min.




Figure 4: Comparison of the CD spectra of human relaxin, human RLF, and porcine relaxin. The spectra of porcine relaxin and RLF are nearly identical.



RLF tracer was prepared from a partially protected intermediate. This procedure allowed for iodination of one or both of the A chain tyrosines without damage to the tryptophan side chain. The different derivatives could be separated by HPLC to yield a carrier-free tracer (Fig. 5). The new factor was tested in the relaxin and insulin receptor assay, and the results depicted in show relaxin rather than insulin-like cross-reactivities. A 100-fold excess of human RLF displaces 50% of the relaxin tracer from a mouse brain relaxin receptor preparation. The difference in affinity is still within the range of specific binding, i.e. several orders of magnitude better than the binding of insulin or guinea pig relaxin to this receptor (13) so that one can conclude that RLF recognizes the relaxin receptor. This result was surprising because the critical Arg-X-X-X-Arg sequence in RLF is offset toward the C-terminal end of the B chain by exactly one turn of the helix (Fig. 1). Although the new factor projects the arginines at nearly right angles away from the molecular surface (as does relaxin(20) ), shifting of the whole receptor-binding site must present quite a different binding environment to the receptor. To prove that the relaxin-like factor competes with relaxin for the relaxin receptor, it was important to determine whether or not relaxin would compete for an RLF-specific receptor if one could be detected. Of the tissues tested with I-RLF as tracer such as brain, uterus, skeletal muscle, kidney, and liver, only the brain and the uterus membrane preparation showed specific binding (Fig. 6). These are tissues that also bind relaxin in a competitive and saturable manner. To test for cross-reactivity the assays were performed with tracers and competitive cold molecules exchanged. The results shown in suggest strongly that RLF does have its own receptor in these tissues and that the relaxin receptor is recognized by RLF, but with a significantly lower affinity than relaxin. Furthermore, the data support the notion that the brain and uterine relaxin receptors differ as concerns cross-reactivity. The uterine relaxin receptor barely recognizes RLF, whereas the brain receptor shows moderate cross-reactivity. In general, the RLF receptor binds its substrate with greater affinity than the relaxin receptor displays toward relaxin.


Figure 5: RLF tracer preparation. Shown is the elution record of the HPLC separation of the reaction mixture. The largest peak is unmodified RLF and the shaded region is the major radioactive peak used as tracer. Chromatography was performed on Aquapore 300 (2.1 30 mm) using a linear gradient from 23% solvent B to 34% solvent B over 60 min (solvent A: 0.1% trifluoroacetic acid in HO and solvent B: 0.1% trifluoroacetic acid in 80% acetonitrile) at a flow rate of 0.1 ml/min.




Figure 6: The tissue distribution of RLF receptors in female estrogen-primed mice as measured in vitro in a receptor-binding assay.



It has been established that binding to the relaxin receptor requires two arginines in an n, n + 4 position on an -helix, and therefore, one can assume that RLF binds the relaxin receptor with the analogous arginine sequence in the n + 4, n + 8 position (relative to relaxin). A mouse bioassay with up to 20 µg of RLF per mouse shows however that the factor by itself does not cause widening of the symphysis pubis (Fig. 7A). If RLF is indeed a hormone instead of an autocrine factor it could in theory act as a competitive inhibitor of relaxin.


Figure 7: A, comparative bioassay of relaxin, RLF, and an optimal dose of both. RLF alone does not cause sympheseal widening, but the high dose of the mixture still improves upon the high dose of relaxin alone. B, bioactivity of an increasing amount of relaxin in the presence and absence of 5 µg of RLF/animal. The increase in sympheseal width was easily recognized. C, bioactivity of an increasing amount of RLF in the presence of a uniform amount of human relaxin again shows relaxin enhancement. hRLX, human relaxin.



To further explore a possible role for RLF, a bioassay was performed using ovariectomized estrogen-primed virgin female mice(14) . Groups of five animals received either human relaxin at a suboptimal dose or a mixture of 0.2, 0.4, and 0.8 µg of human relaxin and 5 µg of relaxin-like protein as given in Fig. 7B. The RLF significantly increases the activity of human relaxin in the mouse bioassay. Increasing RLF concentrations in the presence of 0.5 µg of human relaxin shows that 5 µg of RLF is optimal (Fig. 7C). Again the effect of the relaxin-like factor is clearly recognized. In the next assay relaxin alone, RLF alone, and a maximal dose of both are compared (Fig. 7A). Although RLF alone has no effect, the relaxin effect at maximal dose is still augmented by RLF. In light of the lack of additive effects during receptor binding studies in vitro, this result can be explained only as receptor ``cross-talk,'' i.e. the intact cell is required to show the cooperative effect of RLF on the biological activity of relaxin.

The new factor appears not to be limited to Leydig cells (21) and is certainly not insulin-like as regards functional affinities. Although we have demonstrated relaxin-enhancing properties, it is distinctly possible that the new protein has another independent biological activity as yet to be discovered. At this time the term RLF may be most appropriate.

The competitive character of the relaxin/relaxin-like factor interaction with the mouse brain receptor is surprising when one considers the shift of the receptor interacting region. Comparatively small changes, i.e. substitution of homoarginine or lysine for arginine, reduce receptor binding of relaxin much more (3) than shifting the whole binding region by one helix turn (5.4Å) in relation to the molecular core.

It seems quite implausible that a molecule that binds to an extraneous receptor would not be an inhibitor of the natural ligand of that receptor. Instead of the expected partial inhibition of relaxin by RLF a distinct stimulation of the relaxin response was observed. A similar ``cross-talk'' effect is also observed between relaxin and insulin in the pregnant rat ovarian fat cell assay(22) . The physiological importance of these observations require close scrutiny. Human relaxin is not quite as active in humans as porcine relaxin. The relaxin-like factor may therefore play an important supportive role for the relaxin action in humans. In addition, preliminary experiments suggest that RLF may play a role independent of relaxin in the male gonads.

  
Table: Binding of relaxin and RLF to mouse brain and uterus



FOOTNOTES

*
This work was supported by National Institutes of Health Grant NIHGMS-48829 and National Science Foundation Grant MCB-9406656 and by the Medical University of South Carolina institutional research funds for 1994-1995. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: RLF, relaxin-like factor; Acm, acetamidomethyl; HPLC, high performance liquid chromatography; MBzl, 4-methylbenzyl.


ACKNOWLEDGEMENTS

We thank Barbara Rembiesa and Robert Bracey for their excellent technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.