Biomedical subjects

R J Ryan

Publications and source records attributed to R J Ryan.

At least 19 recordsLinked to original sources

PubMed1992

Inhibition of TSH bio-activity by synthetic beta TSH peptides.

The in vitro bioactivity of the human beta TSH subunit was investigated utilizing eleven overlapping synthetic peptides representing the entire 112 residue sequence. The peptides were tested for both stimulatory and inhibitory activity in two sensitive bioassay systems: the first based on cAMP production in FRTL-5 rat thyroid cells, and the second based on stimulation of iodine trapping by the same continuous cell line. Peptides from three distinct regions of the beta-subunit showed concentration dependent inhibition of TSH bio-activity, including beta 1-15, beta 11-25, beta 31-45, beta 81-95, and beta 91-105 with IC50 values ranging from 150 to 304 microM. An additional peptide representing the entire sequence of the "intercysteine loop" region of beta TSH, beta 31-52, also inhibited TSH activity with somewhat higher potency than its fragment peptide beta 31-45 (IC50 of 87.5 +/- 14.7 microM for beta 31-52 versus 207 +/- 92.4 microM for beta 31-45). Three of these, beta 1-15, beta 31-45, and beta 31-52, also inhibited binding of TSH to the receptor in a radio-receptor assay, as previously reported (1), supporting their importance in receptor interaction. None of the synthetic peptides stimulated either cAMP production or iodine trapping. Two other overlapping peptides, beta 81-95 and beta 91-105, possessed bio-inhibitory activity but did not inhibit binding of labeled TSH. Computer analysis of this sequence predicted an extended turn structure for this region. This region has been referred to as the "determinant loop" as it is bounded by cysteine residues at positions 88 and 95 that many believe form a disulfide bond in the native subunit. The current data suggests the beta 88-95 region may play a role in receptor activation after initial binding of hormone to receptor.

Amino Acid Sequence↗

PubMed1992

The identification of spinal pathology in chronic low back pain using single photon emission computed tomography.

Bone scintigraphy with single photon emission computed tomography (SPECT) offers improved lesion detection and localization when compared to conventional planar imaging. The SPECT findings were investigated in 80 consecutive patients (aged 18-70 years, median 44) referred to a rheumatology outpatient clinic with low back pain persisting for more than 3 months. Lesions of the lumbar spine were demonstrated in 60% of patients using SPECT but in only 35% with planar imaging. Fifty-one per cent of all lesions were only detected by SPECT, and lesions visualized on SPECT could be precisely localized to the vertebral body, or different parts of the posterior elements. Fifty per cent of lesions involved the facetal joints of which almost 60% were identified on SPECT alone. X-rays of the lumbar spine, with posterior oblique views, failed to demonstrate abnormalities corresponding to almost all SPECT posterior element lesions although it identified abnormalities corresponding to over 60% of anterior SPECT lesions. Computed tomography (CT) was performed in 30 patients with a SPECT lesion and sites of facetal joint activity corresponded to facetal osteoarthritis in 82%. It is concluded that bone scintigraphy with SPECT in patients with chronic low back pain demonstrates many lesions not seen with either X-ray or conventional planar imaging. In addition anatomical localization is greatly enhanced with bone SPECT. The technique offers improved diagnosis in a group of patients often difficult to evaluate, and in particular a means of detecting apophyseal joint pathology which may be responsive to treatment.

Adult↗

PubMed1992

Identification of hormone-binding regions of the luteinizing hormone/human chorionic gonadotropin receptor using synthetic peptides.

A comprehensive series of overlapping synthetic peptides have been used to study the relationship between the primary structure of the ovarian receptor for LH/human CG (hCG) and hormone binding. Twenty-four consecutive, overlap peptides that replicate the entire extracellular domain of the rat luteal receptor have been synthesized by standard solid-phase techniques on an automated synthesizer. Eight additional peptides from the extracellular domain and three peptides replicating the putative extracellular loop regions have also been synthesized. Each peptide was evaluated in RRAs for interaction with hCG by measuring its ability to competitively inhibit binding of 125I-hCG to membrane receptor. Twelve peptides were found to be potent in RRAs and caused a reduction of half-maximal binding of 125I-hCG at concentrations of 10-250 x 10(-6) M. The 12 active peptides (and adjacent inactive peptides) defined at least 4 independent receptor regions that can interact with hormone. One site near the NH2-terminus was localized to receptor residues Arg21-Pro38. Two more sites of hormone interaction were identified by peptides replicating residues Arg102-Thr115 and Tyr253-Phe272. A fourth binding region was identified in the third putative extracellular loop, replicated by rat luteal receptor peptide Lys573-Lys583. The amino acid sequences of the four active rat LH/hCG receptor regions were aligned and compared with published sequences for other glycoprotein hormone receptors. Three regions (Arg102-Thr115, Tyr253-Phe272, and Lys573-Lys583) showed high sequence homology with the human LH/hCG receptor, human TSH receptor, and rat FSH receptor and may represent contact sites for the alpha-subunit of hormone. The other binding region, Arg21-Pro38 had low sequence homology with the other glycoprotein hormone receptors and is postulated to be a binding determinant for beta-hCG/LH. This report demonstrates that synthetic overlap peptides of confirmed sequence can be used to successively identify hormone interaction sites of glycoprotein hormone receptors.

Amino Acid Sequence↗

PubMed1991

Further characterization of the receptor-binding region of the thyroid-stimulating hormone alpha subunit: a comprehensive synthetic peptide study of the alpha-subunit 26-46 sequence.

Previously, using a synthetic peptide strategy, we determined that the region of the common glycoprotein hormone alpha subunit between residues 26 and 46 is a site of interaction of the hormone with the thyroid membrane-bound receptor for thyroid-stimulating hormone (TSH). We have undertaken to identify further the specific residues within this 21-amino acid span that are critical in hormone receptor binding. We synthesized three nested sets of peptide, two in which we systematically truncated the amino-terminal region of the sequence and another in which we truncated the carboxyl-terminal region, and we synthesized a fourth nested set in which we systematically substituted alanine for the native residues from the region of highest activity. Each peptide was tested in a TSH radioreceptor assay for its ability to inhibit binding of 125I-labeled bovine TSH to porcine thyroid membranes. Removal either by truncation or alanine substitution, of several specific residues resulted in a significant reduction in the ability of the sequence to interact with receptor; these residues included Cys31, Cys32, Phe33, Arg35, Arg42, Lys44, and Lys45, suggesting that they are crucial for binding activity. Loss of activity also occurred with substitution for Gly30 and Ser34, but the reduction was less pronounced. Amino-terminal truncation of the sequence through Arg35 (leaving the alpha-subunit peptide 36-46) resulted in greater than 98% loss of activity of the sequence. We conclude that two distinct receptor binding regions lie within the alpha-subunit 26-46 sequence. The first lies between residues Gly30 and Arg35 and includes Cys31, Cys32, and Phe33 as important constituents, and the second region lies between residues Arg42 and Lys45 and includes Lys44 as an important residue and Ser43 as a less important component.

Amino Acid Sequence↗

PubMed1991

Residues in the alpha subunit of human choriotropin that are important for interaction with the lutropin receptor.

Synthetic peptides were used to probe the structure-function relationships between human choriotropin (hCG) and the lutropin (LH) receptor. Previously, a peptide region of the alpha subunit of hCG, residues 26-46, had been shown to inhibit binding of 125I-hCG to the LH receptor in rat ovarian membranes (Charlesworth, M.C., McCormick, D.J., Madden, B., and Ryan, R.J. (1987) J. Biol. Chem. 262, 13409-13416). To determine which residues are important for this inhibitory activity, peptides were truncated from either the amino or carboxyl terminus, or individual residues were substituted with alanine. The amino-terminal boundary was determined to be Gly-30 and the carboxyl-terminal boundary, Lys-44. This core peptide contained all the residues needed for full activity of the parent peptide 26-46. Arg-35 and Phe-33 were particularly important residues; when they were substituted with alanine, the peptide inhibitory potencies were decreased. Ser-43, Arg-42, Cys-32, and Cys-31 were also important but to a lesser degree. These results are consistent with predictions based on chemical and enzymatic modification studies and provide insight into which residues are important for interaction between hCG and the LH receptor.

Alanine↗

PubMed1991

The antigenic structure of the human glycoprotein hormone alpha-subunit: III. Solution- and solid-phase mapping using synthetic peptides.

Twenty-seven synthetic peptides, representing the entire structure of the human glycoprotein hormone alpha-subunit were used to map the antigenic structure of the alpha-subunit. Solution phase and solid phase assays were performed with these peptides and a panel of eight monoclonal antibodies (MAb). Two dominant regions were localized between residues 22-37 and 70-87. All eight antibodies recognized these regions, but differed somewhat with respect to whether they saw the more N-terminal, middle, or C-terminal portions of these regions. The sequence of residues 13-22 was recognized by three MAbs. The C-terminal region from residues 84-92 was recognized by three MAbs. All MAbs recognized conformational epitopes in that they reacted with two or more regions. Three MAbs (two against free alpha and one against human CG) have linear amino acid sequences as part of their conformational epitope.

Antibodies, Monoclonal↗

PubMed1990

Inhibition of thyrotropin binding to receptor by synthetic human thyrotropin beta peptides.

In order to study the structure and function relationships of the thyrotropin (TSH)-specific beta-subunit, we produced 11 synthetic overlapping peptides containing the entire 112-amino acid sequence of human beta TSH and tested them for activity in TSH radioreceptor assay using both human and porcine thyroid membranes. Synthetic peptides representing four regions of the beta-subunit demonstrated the ability to inhibit binding of 125I-bovine TSH to crude thyroid membranes. The peptide representing the -COOH terminus of the subunit (beta 101-112) possessed highest binding activity, inhibiting binding of labeled TSH with an EC50 of 80 microM. The remaining active peptides were: beta 71-85 (104 microM), beta 31-45 (186 microM), beta 41-55 (242 microM), and beta 1-15 (331 microM). Specificity of the binding activity was shown by the inability of the peptides representing the remainder of the subunit to inhibit binding of label and by the inability of any of the peptides to inhibit binding of 125I-epidermal growth factor to the same thyroid membranes. The low affinity of the peptides as compared with native hormone is in agreement with previous studies of synthetic alpha-subunit peptides and, further, suggests that the interaction of beta TSH with receptor is multifaceted, requiring cooperative binding of these sites for the observed high affinity of the whole hormone. These studies are in agreement with previous predictions of active regions by chemical modification but add two regions to the list, showing the utility of the synthetic peptide strategy in the study of peptide hormone structure-activity relationships.

Amino Acid Sequence↗

PubMed1990

Synthetic alpha-subunit peptides stimulate testosterone production in vitro by rat Leydig cells.

The glycoprotein hormones (LH, hCG, FSH, and TSH) have a common 92-amino acid alpha-subunit which is noncovalently linked to a hormone-specific beta-subunit. Synthetic peptides of the alpha-subunit have been shown to inhibit binding of [125I]iodo-hCG to rat ovarian membrane and [125I]iodo-TSH to human thyroid membrane preparations. Synthetic overlapping peptides of the alpha-subunit of hCG were prepared by solid phase techniques and tested in a standard in vitro rat Leydig cell bioassay. Three regions in the alpha-subunit (alpha 1-15, alpha 30-45, and alpha 71-85) were found to stimulate testosterone production. All three regions correlate with inhibition of hCG binding to ovarian receptors, but subtle differences exist between the binding sites and effector sites. These data indicate that the glycoprotein alpha-subunit has intrinsic bioactivity.

Amino Acid Sequence↗

PubMed1990

The antigenic structure of the human glycoprotein hormone alpha-subunit. I. Characterization of anti-alpha monoclonal antibodies.

The glycoprotein hormones CG, LH, FSH, and TSH are composed of two noncovalently linked subunits, alpha and beta. The beta-subunit confers hormone specificity, while the alpha-subunit is homologous within a species. To help in determining the antigenic structure of the common alpha-subunit, six monoclonal antibodies (mAbs) to the free or heterodimeric alpha-subunit of human (h) gonadotropic hormones have been prepared and, along with two previously isolated mAbs, have been characterized for binding specificity to alpha- and beta-subunits and the human glycoprotein hormones, CG, LH, FSH, and TSH. Each mAb was derived from hybidomas of FO myeloma cells fused with spleen cells from mice immunized with free alpha-subunit, hCG or hFSH. mAbs A101, A102, and E512 were specific for the alpha-subunit but showed the highest affinity for the intact hormone; K2.18, K94.6, E501, E502, and E511 were specific for free alpha. All of the antibodies inhibited binding of 125I-hCG to luteal membrane receptor, and 125I-labeled mAbs did not recognize hCG/receptor complex. Characterization by two-site binding assays using alpha, hCG, or hFSH as antigen revealed that all the mAbs bind to unique sites on alpha which may be overlapping, and which are modified in the intact hormone. The antigenic sites for mAbs E502, E511, and K2.18 are at least partially linear because they bind to reduced, carboxymethylated alpha.

Animals↗

PubMed1990

The antigenic structure of the human glycoprotein hormone alpha-subunit: II. Cross-species comparisons.

Eight monoclonal antibodies, specific for the glycoprotein hormone alpha-subunit, were raised against human free alpha-subunit, human FSH, or human CG. All of these antihuman monoclonal antibodies were tested for cross-reactivity with alpha-subunits derived from bovine, porcine, equine, bull frog, sea turtle, turkey, and ostrich glycoprotein hormones. All showed cross-reactivity with affinities ranging from 10(-4) to 10(-8) depending upon the antibody and the species of alpha-subunit. Cyanogen bromide fragments of bovine and equine alpha, when tested with selected antibodies indicated that antigenic determinants could be localized in two regions: alpha 9-33 and alpha 76-92. Comparison of amino acid sequences, and relative potencies, suggest that major antigenic determinants involve residues 21, 22, and 23 (F-F-S in human alpha) and 76-85 (G-G-F-K-V-E-N-H-T-A in human alpha). As part of this study the N-terminal amino acid sequences of bull frog, sea turtle, turkey, and ostrich alpha-subunits were determined and reported for the first time.

Amino Acid Sequence↗

PubMed1990

Molecular modeling of residues 38-57 of the beta-subunit of human lutropin.

Several regions on both the alpha- and beta-subunits of human LH comprise the receptor-binding domain of the hormone. One of these, a disulfide loop peptide containing residues 38-57 on the beta-subunit, also stimulated steroidogenesis in rat Leydig cells. Circular dichroism analysis and a Schiffer-Edmundson helical wheel projection of beta-(38-57) revealed the possibility of an amphipathic alpha-helical structure through its N-terminal region. Secondary structure prediction algorithms do not predict alpha-helix in beta-(38-57), but, rather, suggest a sheet-coil-sheet topology. Homology searches between this peptide and proteins with known structure revealed that the two best matches are with prealbumin-(10-30) and melittin-(1-26). Based on hydrophobic moment calculations, we suggest that beta-(38-57) more closely resembles melittin, a known example of an amphipathic helix. Molecular models were constructed that included an alpha-helix between Pro-39 and Pro-50 producing a hydrophilic face involving Thr-40, Arg-43, and Gin-46. Loop closure was performed either visually or by an incremental minimization procedure, using distance constraints to patch in a disulfide bond. Molecular dynamics at 300, 360, and 1000 K were used to explore the local conformational space, and dynamic structures were minimized. The most reasonable structures were found with the 300 and 360 K simulations, with those at 360 K consistently producing structures with lower conformational energies. In each of these simulations, the N-terminus of the alpha-helix unraveled to form a reverse turn (predicted by the GOR algorithm) which include Cys-38, Pro-39, Thr-40, and Met-41. Simulations at 1000 K produced the most variation in structure, but these were deemed unreasonable. Although not all possible conformations were explored, several models were found that comply with the assumption of an amphipathic helix in the N-terminal half of the peptide.

Algorithms↗

PubMed1989

Purification, characterization, and amino-terminal sequence of rat ovarian receptor for luteinizing hormone/human choriogonadotropin.

The luteinizing hormone (LH)/human choriogonadotropin (hCG) receptor of rat ovary was solubilized with Lubrol PX in the presence of 20% glycerol and protease inhibitors, and purified by one-step affinity chromatography. Purified receptor had a specific hCG binding capacity of 4900 pmol/mg protein, and displayed a single class of high affinity binding sites (Ka = 6.20 X 10(9) M-1). An 11,200-fold purification over the starting crude homogenate was achieved. The purified LH/hCG receptor was identified by sodium dodecyl sulfate-gel electrophoresis and silver staining as a single protein of 92 kDa. The ability of the purified 92-kDa protein to specifically bind hormone was demonstrated by electroblotting onto Immobilon P membrane, incubation with 125I-labeled hCG, and autoradiography of the blot. In addition to a 92-kDa band, ligand blotting also yielded a 170-kDa band representing receptor dimer. Covalent cross-linking of hCG, with isotope in either the alpha- or beta-subunit, to membrane-bound receptor produced complexes that contained a single receptor component of approximately 92 kDa. The cross-linking studies indicated that both subunits interact with receptor and also suggested receptor dimer formation. Following sodium dodecyl sulfate-electrophoresis, purified receptor was electroblotted onto polyethylenimine-treated glass fiber filters for direct microsequencing in a gas-phase sequenator. Eleven cycles of sequence analysis yielded the unique sequence: NH2-Arg-Glu-Leu-Ser-Gly-Ser-Leu-XXX-Pro-Glu-Pro-COOH. These results indicate that the rat ovarian LH/hCG receptor is a protein of 92 kDa which can be easily purified in microgram amounts. This study also describes a relatively simple technique for electroblotting and microsequencing that should be applicable to other membrane-bound hormone receptors.

Amino Acid Sequence↗

PubMed1989

Synthetic peptide segments of inhibin alpha- and beta-subunits: preparation and characterization of polyclonal antibodies.

The amino acid sequences of human inhibin alpha-, beta A- and beta B-subunits were analyzed for hydrophilicity and chain flexibility to predict regions that are on the surface of the subunits and, therefore, are potential antigenic sites. Based on these analyses, a total of nine peptides were synthesized, and rabbit antisera against the peptides were prepared. Peptides of the N-terminus (residues 1-16 and 13-24) and region 109-123 of the alpha-subunit produced high titer antibodies. Regions 69-79 and 93-105 of the beta A-subunit and region 93-104 of the beta B-subunit were also immunogenic. Immunoblotting of an inhibin preparation with anti-alpha-peptide antiserum revealed that a 32K band (inhibin) and an 18K band (alpha-subunit) were stained. Immunoblotting with anti-beta-peptide antiserum detected a 32K band (inhibin), a 24K band (activin), and a 14K band (beta-subunit). Injection (iv) of these antisera into rats induced dramatic elevation of serum FSH in 6-12 h and suggested immunoneutralization of endogenous inhibin. RIAs for each subunit were developed using radioiodinated peptides as tracers. Competition binding assays indicated crossreactivity with human follicular fluid, semen, serum, plasma, and crude inhibin preparations. Parallel dilution curves were obtained. Antisera against beta A- and beta B-subunit peptide cross-reacted with each other. In immunocytochemical studies, these antisera were used in conjunction with gold-labeled goat antirabbit immunoglobulin G to localize inhibin in cells of the rat testis. Specific staining of inhibin was localized within the Sertoli cells of some tubules in adult rat testis. Positive staining could be blocked by preadsorbing the sera with the appropriate synthetic peptide. These results suggest that antibodies against synthetic inhibin peptides are useful in elucidating the roles of inhibin and activin.

Animals↗

PubMed1989

Role of the beta 93-100 determinant loop sequence in receptor binding and biological activity of human luteinizing hormone and chorionic gonadotropin.

The intercysteine loop sequence (93-100) in the beta-subunit has been postulated to be important for receptor binding and specificity in the glycoprotein hormones, LH and human CG (hCG). To demonstrate this directly, and to characterize the structural features essential for activity, we prepared a series of synthetic peptides and analogs incorporating this determinant loop region. Peptides were assayed for inhibition of labeled hCG binding to ovarian membrane receptors and stimulation of testosterone production in Leydig cells. Peptides with the native (93-100) sequence from hCG and hLH inhibited hCG binding half maximally at 2.18 and 2.62 x 10(-4) M, respectively, while the sequence from FSH was inactive. Isosteric substitution of Ala for Cys resulted in an inactive peptide, indicating that the (93-100) disulfide bridge is essential for activity. Optimal binding activity requires at least one net positive charge among the side chains, as shown by loss of activity in hybrid analogs with neutral or negative charges conferred by progressive replacement of Arg by Asp at 94 and 95 or by introduction of Asp at 96 and 97. Despite binding to receptors, the native sequence did not promote testosterone production at doses up to 10(-2) M. This contrasts with a second receptor binding sequence, beta (38-57) that activates testosterone production. There are differences between the (93-100) and (38-57) loop sequences in their chemical and physical properties, biological activity and antigenicity. While the cumulative evidence suggests that they associate with counterpart sites in alpha-subunit to form a topographical binding domain in the whole hormone, our results suggest that each sequence may contribute in different ways to activation of postreceptor events.

Amino Acid Sequence↗

PubMed1988

Primary and secondary structural determinants in the receptor binding sequence beta-(38-57) from human luteinizing hormone.

The intercysteine "loop" sequence 38-57 in the beta subunit has been shown to be a determinant for expression of biological activity in human lutropin (hLH) and choriogonadotropin (hCG) [Keutmann, H. T., Charlesworth, M. C., Mason, K. A., Ostrea, T., Johnson, L., & Ryan, R. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 2038]. Together with other sequences, the 38-57 region may contribute to a multicomponent receptor binding domain in hLH/hCG. Because the structural features influencing activity in this important region are not easy to evaluate in the full-length subunit, we have used analogues of hLH beta-(38-57) prepared by solid-phase synthesis. The peptides were tested for inhibition of 125I-labeled hCG binding to rat ovarian membrane receptors. Secondary structure was analyzed by circular dichroism (CD) and by reactivity with antibodies to the native 38-57 peptide. An analogue lacking the 38-57 disulfide linkage retained 20% receptor binding and full immunoreactivity. "Far"-ultraviolet CD profiles were essentially identical with those of the disulfide-intact peptide; a transition from 10% to 30% alpha-helix in 90% trifluoroethanol was characteristic of both. The peptide thus appears not to require the disulfide bridge to retain a looped conformation with amphipathic secondary structure. An essential positive charge at position 43 was shown by complete loss of activity upon substitution of Asp or Ala for the Arg found in all known species of LH. Other analogues showed a requirement for a neutral residue at position 47, also highly conserved.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

The effect of deglycosylated human chorionic gonadotropin on corpora luteal function in healthy women.

Fourteen healthy young women were studied through a control and a treatment menstrual cycle in two series of experiments. In the first series, they were given one of four doses of deglycosylated human chorionic gonadotropin (hCG) as a 24-hour infusion during the mid-luteal phase of the cycle. In these studies, there were no significant alterations of the length of the luteal phase of the treatment cycle, and there was no decrease in serum progesterone (P) during the infusion. In fact, serum P increased during the infusion. In the second series of studies, five subjects were given a 48-hour infusion of normal saline during the control cycle, and a 48-hour infusion of deglycosylated alpha-intact beta-hCG during the treatment cycle, both being administered during the mid-luteal phase. Treatment did not alter luteal phase duration and, again, increased serum P. It is concluded that deglycosylated preparations of hCG are not clinically useful as luteinizing hormone antagonists, probably because of residual agonist activity.

Adult↗

PubMed1988

The glycoprotein hormones: recent studies of structure-function relationships.

The structural features of the heterodimeric glycoprotein hormones (LH, FSH, TSH, and hCG) are briefly reviewed. Removal of carbohydrate chains does not reduce binding of the hormones to membrane receptors, but markedly reduces biological responses. The glycopeptides from the hormone do not reduce binding of native hormone to receptors but do reduce biological responses. Newer data concerned with replication of different regions of the peptide chains of these molecules using synthetic peptides are reviewed and presented. These studies indicate that two regions on the common alpha subunit are involved with receptor binding of the LH, hCG, and TSH molecules. These regions are alpha 26 to 46 and alpha 75-92. Two synthetic disulfide loop peptides from the hCG beta subunit beta 38-57 and beta 93-100 also block binding of hCG to its receptor. In addition, the beta 38-57 peptide stimulates testosterone production by Leydig cells. These data indicate that glycoprotein hormone binding to plasma membrane receptors involves a discontinuous site on the hormone that spans both the alpha and beta subunits, and that the alpha subunit sites are similar for several hormones.

Amino Acid Sequence↗

PubMed1988

The effects of synthetic alpha-subunit peptides on thyrotropin interaction with its receptor.

Synthetic peptides of the alpha-subunit of human glycoprotein hormones have been shown previously to inhibit binding of [125I]iodo-hCG to ovarian membranes, thus indicating the importance of the alpha-subunit in the structure-function relationships of the gonadotropic hormone. These same synthetic alpha-subunit peptides, the sequences of which are common to all human glycoprotein hormones, were found to inhibit the binding of [125I]iodo-TSH to human thyroid membrane preparations and FRTL-5 rat thyroid cells. The active portions of the subunit were represented in synthetic peptides alpha 21-35, alpha 31-45, alpha 26-46, and alpha 81-92, indicating that 2 separate sites within the alpha-subunit have binding activity for TSH. Peptides alpha 26-46 and alpha 31-45 were also found to potently inhibit the stimulation of adenylate cyclase activity by bovine TSH in TSH bioassay using FRTL-5 cells. Seven other synthetic peptides, including the remainder of the 92-amino acid sequence of the alpha-subunit, demonstrated little or no ability to inhibit binding of the tracer or inhibit the bioactivity of intact TSH. The findings were very similar to those of previous studies involving hCG binding, except that the two active sites appeared to be somewhat shifted towards the COOH-terminal end of the subunit. These studies support the concept of the importance of the alpha-subunit in receptor binding of all glycoprotein hormones and demonstrate the utility of the overlapping synthetic peptide strategy in investigations of protein structure-function relationships.

Amino Acid Sequence↗