GREAT/LGR8 Is the Only Receptor for Insulin-Like 3 Peptide

Natalia V. Bogatcheva, Anne Truong, Shu Feng, Wolfgang Engel, Ibrahim M. Adham and Alexander I. Agoulnik

Department of Obstetrics and Gynecology (N.V.B., A.T., S.F., A.I.A.), Baylor College of Medicine, Houston, Texas 77030; and Institute of Human Genetics (W.E., I.M.A.), University of Göttingen, D-37073 Göttingen, Germany

Address all correspondence and requests for reprints to: Dr. Alexander I. Agoulnik, Department of Obstetrics and Gynecology, 6550 Fannin Street, Baylor College of Medicine, Houston, Texas 77030. E-mail: agoulnik{at}bcm.tmc.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
During male development testes descend from their embryonic intraabdominal position into the scrotum. Two genes, encoding the insulin-like 3 peptide (INSL3) and the GREAT/LGR8 G protein-coupled receptor, control the differentiation of gubernaculum, the caudal genitoinguinal ligament critical for testicular descent. It was established that the INSL3 peptide activates GREAT/LGR8 receptor in vitro. Mutations of Insl3 or Great cause cryptorchidism (undescended testes) in mice. Overexpression of the transgenic Insl3 causes male-like gubernaculum differentiation, ovarian descent into lower abdominal position, and reduced fertility in females. To address the question whether Great deletion complements the mutant female phenotype caused by the Insl3 overexpression, we have produced Insl3 transgenic mice deficient for Great. Such females had a wild-type phenotype, demonstrating that Great was the only cognate receptor for Insl3 in vivo. We have established that pancreatic HIT cells, transfected with the INSL3 cDNA, produce functionally active peptide. Analysis of five INSL3 mutant variants detected in cryptorchid patients showed that P49S substitution renders functionally compromised peptide. Therefore, mutations in INSL3 might contribute to the etiology of cryptorchidism. We have also showed that synthetic insulin-like peptides (INSL4 and INSL6) were unable to activate LGR7 or GREAT/LGR8.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN MAMMALS, BOTH undifferentiated ovaries and testes are located originally in the abdominal position. During male development, testes descend from this initial position to the scrotum. Two phases of testicular descent have been defined (1). First, the transabdominal phase occurs between 10 and 15 wk of gestation in human embryos and between d 15.5 and d 17.5 post coitum in mouse embryos. An outgrowth of caudal genitoinguinal ligament, gubernaculum, and a regression of cranial suspensory ligament direct movement of the testes toward the inguinal region. Second, the inguinoscrotal phase of testicular descent occurs in humans before birth and in mice within the first 20 d of neonatal development. During this stage, shortening of the gubernacular cord and swelling of the gubernacular bulb provide a passage for the testes into the scrotum. Recently, it was established that two genes encoding the insulin-like 3 peptide (INSL3) and the GREAT (also called LGR8) G protein-coupled receptor (GPCR) control development of gubernaculum. Male mice deficient for Insl3 (2, 3) or for Great (4, 5) exhibit identical high abdominal cryptorchidism. In both cases, gubernaculae fail to differentiate. In males, Insl3 is produced in pre- and postnatal Leydig cells of testes (6). Expression of the Great gene is detected in several organs, with the highest expression level in gubernaculae, testes, and brain (4, 5). Based on the similarity of the mutant male phenotype, we have suggested that the products of the two genes could, in fact, function as a cognate ligand-receptor pair during development (4, 5).

It was shown that synthetic INSL3 peptide (7) activates GREAT receptor in vitro. Closely related to INSL3, relaxin peptide also activates GREAT, as well as another GPCR, LGR7 (8, 9); whereas synthetic INSL3 fails to stimulate LGR7 receptor in vitro (7). GREAT and LGR7 receptors exhibit a high degree of homology and belong to the same subfamily of GPCRs as the glycoprotein hormone receptors (4, 5, 8, 9). Expression of two receptors overlaps in several tissues. Both LGR7 and GREAT respond to ligand stimulation through a cAMP-dependent pathway, distinct from that of the structurally related insulin and insulin-like growth factors. The indiscriminate character of relaxin interaction with both LGR7 and GREAT raises the question of specificity of hormone-receptor pairing in the relaxin-like group of peptides. To ascertain the possibility of redundancy of receptors for the INSL3, we designed the study where we examined the role of GREAT in the INSL3 signaling in vivo. It was shown earlier that transgenic overexpression of the Insl3 in females results in outgrowth of gubernaculae and descent of the ovaries into the low intraabdominal position (10). Here we demonstrate that the Great deletion rescues the phenotype caused by the Insl3 overexpression, indicating that the Great receptor is the only receptor for Insl3.

We have shown previously, that a unique T222P substitution in GREAT detected in a cryptorchid patient renders a nonfunctional protein, unable to respond to the ligand stimulation (5). Mutation analysis of the INSL3 gene in cryptorchid patients also revealed several unique variants with the single-amino acid substitutions (11, 12, 13, 14, 15, 16, 17, 18). Functional significance of these variants remained unclear to date. Our results indicate that mutant INSL3 (P49S) peptide in one of the cryptorchid patients fails to activate the GREAT receptor. Thus, mutation in INSL3 can be causative in the development of the cryptorchid phenotype in humans.

We have also demonstrated that whereas synthetic INSL3 peptide activates GREAT receptor in vitro, synthetic INSL4 and INSL6 remain ineffective. Together, these results demonstrate an exclusive role of INSL3 and GREAT in testicular descent.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Complementation of the Abnormal Phenotype in Insl3 Transgenic Mice Deficient for Great
To address the question whether Great is the only receptor for Insl3, we have produced transgenic mice overexpressing Insl3 and deficient for Great. The phenotypes of the resulting animals are shown in Fig. 1Go. Insl3 transgenic females with a functional copy of Great (Tg(Ins2-Insl3) Great-/+) had the mutant phenotype with ovaries located in the inguinal, low abdominal region. Analysis of the gonadal position in Tg(Ins2-Insl3) Great-/Great- females revealed that Insl3 did not stimulate male-like differentiation of gubernaculae in the absence of the Great receptor (Fig. 1Go). Transgenic females overexpressing Insl3 with the deletion of Great had a wild-type phenotype, with ovaries in the normal, high abdominal position; males of the same genotype developed cryptorchidism. Expression of Lgr7, related to the Great receptor, was clearly detected in the gubernaculum (data not shown). Despite this, Insl3 could not stimulate gubernacular development in the absence of Great.



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Fig. 1. Insl3 Fails to Stimulate Gubernacular Differentiation in Mice with Deletion of the Great Receptor

Phenotypes of the mice with transgenic allele of Insl3, Tg(Ins2-Insl3), with or without functional Great allele. Overexpression of the Insl3 transgene causes differentiation of gubernaculae in females and descent of the ovaries to the inguinal region (1 ). Overexpression of Insl3 fails to stimulate differentiation of the gubernaculum in females with the deletion of the Great receptor; they have a wild-type phenotype (2 ). Insl3 transgenic males have a wild-type phenotype (3 ). Deletion of Great in Insl3 transgenic males causes high intraabdominal cryptorchidism (4 ). b, Bladder; k, kidney; o, ovary; u, uterus; t, testis. Schematic position of the gonads is shown at the bottom of the figure.

 
The other phenotype associated with Insl3 overexpression is a reduced fertility of the mutant females. It was shown, that in Tg(Ins2-Insl3) females there was a loss of embryos due to a fetal mortality during midgestation (10). We have compared fertility of the females with or without Tg (Ins2-Insl3) transgene (Table 1Go). Tg(Ins2-Insl3) transgenic females with a functional allele of Great have reduced fertility as described previously (10). Comparison of the litter sizes of the females deficient for Great, with or without Insl3 transgene, revealed that the two groups have the same fertility (Table 1Go). Thus, deletion of the Great receptor fully complements deleterious effects of the Insl3 hormone overexpression on female gubernaculae differentiation and fertility.


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Table 1. Deletion of the Great Receptor Normalizes Fertility in Ins13 Transgenic Females

 
Production of Recombinant Human INSL3 in HIT Cells
Previously, it has been shown that ACTH-secreting cells AtT20, transfected with relaxin 3 (RLN3) cDNA, produce physiologically active RLN3 peptide (19). In our experiments we used a pancreatic cell line (HIT) to produce recombinant INSL3 peptide and to analyze the ability of this peptide to activate GREAT receptor. HIT cells were transiently transfected with INSL3 cDNA expression construct. Conditioned culture media from these cells containing secreted hormone were used to treat human embryonic kidney 293T cells, transfected with human GREAT or LGR7 cDNA. It has been established that an activation of the GREAT or LGR7 receptors causes an increase of intracellular cAMP (7, 8). We used such an approach to determine the biological activity of the recombinant peptides. Concentration of the INSL3 peptide in the conditioned medium was determined using INSL3-specific RIA. Figure 2Go shows that medium from the HIT cells transfected with a vector DNA did not induce an increase in cAMP concentration in cells expressing GREAT or LGR7. Conversely, medium containing recombinant wild-type INSL3 was able to increase the cAMP level in a dose-dependent manner in cells expressing GREAT. The INSL3-induced activation was specific for the GREAT receptor and did not affect the concentration of cAMP in cells expressing the LGR7 receptor (Fig. 2Go).



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Fig. 2. Recombinant INSL3 Peptide Stimulates GREAT Receptor

Human recombinant INSL3 was produced by transfecting HIT cells with INSL3 cDNA expression vector. INSL3 hormone content in the conditioned medium was measured with RIA; the medium was used to stimulate 293T cells expressing GREAT (circles) or LGR7 (inverted triangles). The equivalent amounts of medium from HIT cells, transfected with vector DNA, were used as a control (squares and triangles, respectively).

 
Analysis of INSL3 Mutant Variants Associated with Cryptorchidism
Several single-base pair variants of the INSL3 gene have been found in human populations. To elucidate the physiological relevance of such substitutions, we analyzed the ability of mutant INSL3 peptides to activate the GREAT receptor. Four mutant variants of INSL3 (P49S, P93L, R102C, and N110K, where the first amino acid corresponds to the first codon in the full-length INSL3 cDNA) previously detected in cryptorchid patients (13, 15, 16, 18) and a unique variant (R102H) found in a normal female (16) have been produced using site-specific mutagenesis. Expression constructs of the INSL3 cDNAs encoding different mutant peptides were introduced into HIT cells. All five constructs produced RNA transcripts of the correct size as evident from the Northern blot analysis (data not shown). The concentration of INSL3 peptides in the conditioned media was determined by RIA, and normalized amounts of media were used to stimulate 293T cells expressing the GREAT receptor. Analysis of cAMP level in INSL3-treated cells revealed that the INSL3 peptide carrying the P49S substitution drastically loses its ability to activate the receptor. R102C substitution only moderately affected INSL3 physiological activity. Neither P93L, R102H, nor N110K substitutions changed the ability of INSL3 to induce cAMP increase in the cells expressing GREAT (Fig. 3Go).



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Fig. 3. Functional Analysis of Mutant INSL3 Peptides

A, 293T cells, transfected with GREAT DNA, were challenged with conditioned media from HIT cells, expressing wild-type INSL3 (squares), P93L mutant (circles), or R102C mutant (triangles). B, 293T cells, transfected with GREAT DNA, were challenged with conditioned media from HIT cells, expressing wild-type INSL3 (squares), P49S mutant (circles), R102H mutant (triangles), or N110K mutant (inverted triangles). INSL3 hormone content in HIT media was measured with RIA. Figure shows the results of one of three representative experiments. Each point represents the mean values with a SE of the duplicate measurements.

 
INSL4 and INSL6 Peptides Do Not Activate LGR7 or GREAT Receptors
As was shown before (7, 8), the GREAT receptor can be activated by both INSL3 and relaxin in vitro. We have studied GREAT activation by two other recently identified relaxin-like peptides, INSL4 and INSL6 (20, 21, 22). Receptors and signaling pathways for these insulin-like peptides are unknown. Based on the homology between insulin gene family members, the predicted structure of the A and B chains of INSL4 and INSL6 has been defined (20, 21, 22). We used synthetic INSL3, INSL4, and INSL6 peptides with the structure depicted in Fig. 4Go to analyze their ability to activate GREAT and LGR7. As shown in Fig. 5Go, synthetic human INSL3 increased intracellular cAMP level in 293T cells expressing GREAT; however, the activity of such peptide was significantly lower than that of recombinant INSL3 (Fig. 3Go) or porcine relaxin (data not shown). Synthetic INSL4 and INSL6 were unable to activate GREAT receptor (Fig. 5Go). None of the used insulin-like peptides was able to activate LGR7 (data not shown). The results demonstrate that synthetic INSL4 and INSL6 peptides do not activate GREAT or LGR7.



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Fig. 4. Primary Structure of Synthetic INSL3 (A), INSL4 (B), and INSL6 (C) Peptides Used in the Experiments

 


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Fig. 5. Stimulation of Cellular cAMP Production by the Synthetic INSL3

Synthetic INSL3 (squares) stimulates dose-dependent cAMP production in 293T cells, transfected with the GREAT cDNA, whereas INSL4 (circles) and INSL6 (triangles) do not. Figure shows the results of one of two representative experiments. Each point represents the mean values with a SE of the duplicate measurements.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cryptorchidism in humans is one of the most frequent congenital abnormalities. Despite clear indications of the hereditary component in the etiology of the disease, the genetic basis of testicular descent remains vague. Using gene targeting and transgenic approaches, our laboratories recently established that the Insl3 hormone and G protein-coupled receptor named Great control the first phase of testicular descent in mice (2, 5, 10). It has been shown that transgenic overexpression of Insl3 in female mice causes male-like differentiation of the gubernaculum and ovarian descent into the inguinal region (10). Transgenic females carrying the Insl3 gene under the control of the rat insulin 2 promoter (Ins2-Insl3) display bilateral inguinal hernia and reduced fertility; transgenic males have a wild-type phenotype. Thus, Insl3 alone controls the first intraabdominal stage of testicular descent in an androgen-independent fashion (10). It was established that both INSL3 and the closely related peptide, relaxin, are capable of activating GREAT receptor in vitro (7, 8). To evaluate the role of the Great receptor in the Insl3-mediated signaling in vivo, we produced mice that carried the Insl3 transgene and a null allele of Great. If Great is the only receptor for Insl3, overexpression of the transgene in mice with a Great deletion will fail to induce gubernacular differentiation in both males and females and, also, restore fertility of the Tg(Ins2-Insl3) Great-/- females. If Insl3 signals through additional unknown receptor(s), the mutant phenotype caused by transgene overexpression will still be present in Great-deficient animals. Analysis of the gonadal position and female fertility in double-mutant animals clearly showed that the first assumption was correct. Deletion of the Great gene completely abrogated the abnormal phenotype associated with the overexpression of Insl3. Therefore, our data prove that the Great receptor is the only receptor responsible for the Insl3-mediated signaling in vivo.

Next, we demonstrated that the production of biologically active INSL3 can be achieved in cultured cells transfected with the INSL3 cDNA. It is generally recognized that members of the insulin superfamily are synthesized as preprohormones; cleavage of signal peptide followed by formation of disulfide bridges and C-peptide exclusion yields the mature hormone. It has been shown previously that the INSL3 peptide produced in vivo from the transgenic allele under rat insulin 2 promoter in ß-cells of pancreas is fully functional and complements the genetic deficiency of the endogenous alleles (10). Because the mode of the pro-INSL3 processing is believed to be similar to that of relaxin and insulin (23), we used an insulin-producing pancreatic ß-cell line (HIT) to generate recombinant INSL3 and to confirm that such recombinant peptide can stimulate the GREAT receptor in vitro. Our results indicate that INSL3 is converted to a mature biologically active form in HIT cells and secreted in the culture medium.

Given the significance of the INSL3-GREAT pathway in testicular descent, the functional analysis of naturally occurring mutations in the INSL3 gene was the next subject of our study. Mutation analysis of the INSL3 gene in almost 600 cryptorchid patients has been reported in several published studies (11, 12, 13, 14, 15, 16, 17, 18). Some of the described variants of INSL3 were found both in cryptorchid patients and in a control population of healthy males and, therefore, most likely represent functionally active hormones. Five INSL3 variants (P49S, P93L, R102C, N110K, and R73X) were found only in the cryptorchid patients (13, 15, 16, 18), whereas R102H substitution was found in a single control female, which, obviously, could not exert the mutant phenotype. Whereas nonsense mutation (R73X) obviously produces a defective peptide, the functional significance of other single-amino acid substitutions was not clear. These five missense mutations can be subdivided into three groups according to the location in pre-proINSL3 structure. The P49S and N110K substitutions are located in the B-chain and A-chain of mature hormone, respectively, whereas P93L and R102C/H are located within the C peptide, excluded from the mature hormone structure. As our data show, only P49S substitution severely reduces INSL3 ability to activate GREAT. A mutated amino acid residue is located in the C terminus of B chain within a highly conserved region of INSL3 (16). All INSL3 cDNA isolated from different mammalian species contain proline in the position corresponding to P49 in the human INSL3 (24, 25). Interestingly, proline 49 is located within two residues from tryptophan 51, which is critical for the receptor binding (26). Proline 49 is believed to be crucial for the proper orientation of tryptophan 51 because its substitution with D-proline reduces receptor binding 25-fold (26). Thus, the P49S mutation probably affects INSL3 interaction with the receptor.

The P93L substitution, located in the middle of C peptide, leads to a change of less conservative residues (13, 25). Taking into account that horse and rodent INSL3 peptides also carry leucine at the corresponding position, it is not surprising that the P93L mutant effectively activates GREAT. The R102C and R102H substitutions are located at the very end of C peptide in front of the endopeptidase cleavage site. Several mammalian INSL3 peptides contain histidine at the same position. Whereas arginine to histidine substitution does not alter the charge of the region, arginine to cysteine mutation could affect the stretch of positively charged residues necessary for the C peptide cleavage (15). Therefore, decreased efficiency of the R102C variant processing might account for the slight reduction of R102C peptide physiological activity found in our experiments. Despite the fact that N110 is conserved in all known INSL3 peptides, N110K substitution does not change the ability of INSL3 to activate GREAT. In summary, we have shown that at least one substitution (P49S) compromises INSL3 physiological activity, and, therefore, could be responsible for the undescended testes phenotype. Other INSL3 variants do not significantly alter the activation properties of the peptide. It should be noted, however, that other characteristics of the mutant peptides, such as cell-specific efficiency of transcription, translation, processing of the mature protein, or its stability, have not been analyzed in the current study. Nevertheless, taking into account a low frequency of the detected mutations in INSL3, we suggest that alterations in this gene could be responsible for only a small portion of the disease cases in humans.

Considering the promiscuity of the GREAT toward relaxin and INSL3 in the in vitro experiments, we analyzed the ability of other related peptides to activate GREAT. It has been reported that recently discovered relaxin 3 peptide (also called INSL7) does not stimulate GREAT receptor but was able to activate relaxin receptor LGR7 (27). Other two members (INSL4 and INSL6) of the relaxin subfamily have been identified recently (20, 21, 22). Expression patterns of INSL4 and INSL6 indicate that these peptides, together with INSL3 and relaxin, could be involved in the regulation of reproductive function in mammals. INLS4 (also known as early placenta insulin-like peptide or placentin) is abundant in the placenta (20). INSL6 is found mainly in the testes, specifically within the seminiferous tubules in spermatocytes and round spermatids (22). The endopeptidases cleavage sites in the INSL4 and INSL6 preprohormone sequences were predicted based on the homology with other members of the insulin/relaxin superfamily (20, 21, 22). We have demonstrated that neither INSL4 nor INSL6 peptides with the predicted structure activate GREAT receptor in our experiments. However, the inability to check the bioactivity of these peptides due to the lack of data concerning their cognate receptors and signaling cascades may require further experiments to prove this conclusion.

In summary, we have demonstrated that the GREAT receptor is the only cognate receptor for INSL3 in vivo. Identification of the specific interaction of the INSL3 with GREAT expands our understanding of the mechanism of testicular descent and cryptorchidism. We have developed an in vitro protocol to assess the physiological significance of different INSL3 mutations. Using this method, we have shown that the P49S mutation of INSL3, detected in the cryptorchid patient, renders functionally compromised peptide and therefore can be accountable for the development of the disease phenotype in the affected carrier.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mouse Breeding and Genotyping
Production and characterization of Tg(Ins2-Insl3) transgenic mice (10) and mice deficient for Great were described previously (4, 5). Tg(Ins2-Insl3) transgenic females were crossed to Greatko/+ heterozygous males, and the resulting Tg(Ins2-Insl3) Greatko/+ were crossed to crsp/crsp mice to obtain animals of the Tg(Ins2-Insl3) Greatko/crsp- genotype. Crsp mice have a complete deletion of the Great gene (4). Greatko/+ heterozygous animals used in this study were the eighth backcross generation of the Greatko knockout allele onto the FVB inbred strain. Both Tg(Ins2-Insl3) transgenic mice and crsp/crsp mutants are coisogenic mutants generated and maintained on the FVB inbred background. The genotype of the mice was identified based on the PCR assays with primers specific for Tg(Ins2-Insl3) (10), Greatko (5), and crsp (4). Estimations of female fertility have been performed on 2- to 3-month-old females of different genotypes derived from the same litters. The t test was used to determine the significance of differences in litter size. The animal studies were approved by Baylor College of Medicine Institutional Committee on animal care.

Production of the cDNA Expression Constructs
Full-length wild-type INSL3 cDNA was amplified by RT-PCR from human testis RNA with primers 5'-(CCCAAGCTT)CCACCATGGACCCCCGT-3' and 5'-(CCCAGATCT)GTAGGGACAGAGGGTCAGCA-3'. The resultant cDNA was subcloned into HindIII/BamHI sites of the eukaryotic cell expression vector pcDNA3.1/myc-HisB (Invitrogen, San Diego, CA). Plasmids were purified using the Concert Midi-prep plasmid preparation kit (Life Technologies, Inc., Gaithersburg, MD). The sequence of the construct was verified by sequencing of both strands using gene-specific and vector-derived primers. To produce targeted mutations in the wild-type INSL3 cDNA we used the QuikChange Site-Directed Mutagenesis kit from Stratagene (La Jolla, CA). Resultant cDNA constructs were verified by sequencing of both DNA strands and recloned into pcDNA3.1/myc-HisB vector. GREAT cDNA expression vector was obtained previously (5). LGR7 cDNA was kindly provided by Dr. A. J. W. Hsueh (7).

Activation of the GREAT and LGR7 Receptors
Porcine relaxin was kindly provided by Dr. O. D. Sherwood, University of Illinois. Synthetic INSL3, INSL4, and INSL6 were obtained from Phoenix Pharmaceuticals, Inc. (Belmont, CA). According to the manufacturer, the peptides were synthesized using a previously described method (28); the polypeptide chain synthesis and disulfide bond coupling were verified by mass spectral analysis.

Recombinant wild-type and mutant INSL3 peptides were obtained by transfecting pancreatic HIT cells grown in a T-25 flask with 5 µg of the expression construct encoding corresponding peptide using Fugene 6 (Roche, Indianapolis, IN). Medium from cells transfected with pCR3.1 vector was used as a control. The exact concentration of INSL3 peptides in the media was assessed with the INSL3 RIA kit (Phoenix Pharmaceuticals, Inc.) utilizing rabbit polyclonal anti-INSL3 serum raised against full-length synthetic INSL3 peptide.

Activation of the LGR7 and GREAT receptors was assayed as described previously (5). 293T cells grown in 24 wells were transfected with approximately 0.5 µg/well of LGR7 or GREAT construct. After 24 h the efficiency of transfection was estimated by the analysis of the secreted AP activity (pAPtag-5 vector from GenHunter, Nashville, TN, was used for cotransfection) with p-nitrophenyl phosphate as a substrate (Sigma Chemical Co., St. Louis, MO). 293T cells were treated with porcine relaxin, synthetic INSLs, or with the HIT conditioned media containing recombinant peptides, in the presence of 250 µM isobutylmethylxanthine for 30 min. Cells were harvested, washed, and lysed with cAMP extraction buffer (Amersham Pharmacia Biotech, Arlington Heights, IL). cAMP level was detected using Amersham enzyme immunoassay system (Amersham Pharmacia Biotech). cAMP concentration in each well was measured in duplicate. All experiments were repeated several times using cells from independent transfections.


    ACKNOWLEDGMENTS
 
We wish to thank Dr. I. Agoulnik for critical comments, Dr. O. D. Sherwood for the kind gift of porcine relaxin, and Dr. A. J. W. Hsueh for LGR7 expression vector.


    FOOTNOTES
 
This work was supported by NIH Grants R01 HD37067 and P01 HD36289, and a grant from the March of Dimes Birth Defect Foundation (to A.I.A.).

Abbreviations: GPCR, G protein-coupled receptor; GREAT, GPCR affecting testis descent; INSL3, insulin-like 3 peptide; LGR, leucine-rich repeat-containing GPCR.

Received for publication March 19, 2003. Accepted for publication August 13, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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