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Although the atrial free wall is a thin structure, atrial depolarization has been shown to have aspects of three-dimensionality. This study asks whether the same is true for atrial repolarization. By using a multi-element dual-surface probe, monophasic action potentials (MAPs) were recorded simultaneously at several opposing sites on the right atrial endocardial and epicardial surfaces in six open-chest pigs. The times of depolarization and repolarization were marked in recordings during sinus, paced rhythms, and during infusions of cold saline to the epicardial surface, which generated a temperature gradient across the atrial wall. Repolarization times were similar on endocardial and epicardial surfaces in some cites, but others showed significant differences during sinus and paced rhythms. Cold saline infusion produced a significant lengthening of MAP duration, and this was more pronouced on the atrial endocardial sites than on the epicardial sites. The observed differences in endocardial and epicardial repolarization times may be due to the presence of atrial pectinate muscles on the endocardial surface. These results suggest that in some regions atrial repolarization is a three-dimensional process. Possible limitations of this study include the fact that the depth of view of MAPs recorded from the atrial wall may extend to the opposing surface.

Humanstructure interaction in vertical vibrations B. R. Ellis, BSc, PhD, CEng, MIStructE and T. Ji, BSc, MSc, PhD, C. Eng, Mistruct E. s This paper deals with the subject of humanstructure interaction in vertical vibrations. It presents some results recorded on a cantilevered grandstand at Twickenham which showed that, although the empty structure had a clearly defined fundamental mode, two modes of vibration were apparent when the spectators were seated. The frequency of the empty stand was between the two frequencies of the occupied stand and a significant increase in damping was noted when the crowd was present...

The luteinizing hormone/chorionic gonadotropin receptor is a member of the seven-transmembrane receptor family. It is coupled, presumably via Gs and Gq, to two signal pathways involving adenylyl cyclase/cAMP and phospholipase C/inositol phosphate (IP). Little is known about the events prior to G-protein coupling: for example, whether these signals are generated from a single or multiple independent origins and mechanisms, when and where they diverge, and how they are transduced. We report novel observations that the cAMP signal and the IP signal originate and diverge upstream of G-protein coupling. The generation of these two signals independently involves Lys583 in exoloop 3 of the rat receptor. For this study, Lys583 of the receptor was substituted with a panel of amino acids, and mutant receptors were assayed for hormone binding and induction of cAMP, inositol monophosphate, inositol bisphosphate, and inositol trisphosphate. No substitutions for Lys583 were permissible for cAMP induction, despite successful surface expression and hormone binding. In contrast, several substitutions were permissible for IP induction. Our results suggest two distinct transmembrane signal conductors for cAMP and inositol phosphate signals and imply particular models of receptor activation not previously suggested.

Human choriogonadotropin (hCG) consists of an alpha subunit and a beta subunit. The existing evidence from various studies using truncation, substitution, synthetic hormone peptides, and hCG crystals suggests that the C-terminal region of the alpha subunit contacts the luteinizing hormone/choriogonoadotropin (LH/CG) receptor and is involved in receptor activation. Despite a deluge of the speculation and the important role of the alpha C-terminal region, direct evidence for its interaction with the receptor has been elusive. Because of the significant biological activity, it is imperative to prove the interaction of the alpha C-terminal region. For this purpose, decamer peptides corresponding to the alpha subunit sequence from His83 to Ser92 (alpha 83-92) were derivatized with the N-hydroxysuccinimide ester of 4-azidobenzoylglycine (ABG) and radioiodinated. The resulting ABG-125I-alpha 83-92 was capable of binding and activating the LH/CG receptor. Furthermore, UV-sensitive ABG-125I-alpha 83-92 exclusively photoaffinity-labeled an approximately of 86-kDa molecule. This labeled molecule was shown to be the LH/CG receptor by various methods including immunoprecipitation by anti-LH/CG receptor antiserum. In addition, evidence is presented that the amino group of alpha Lys91 of alpha 83-92 is in such close proximity to a carboxyl group of the receptor that this pair is cross-linked to form an amide, a zero length cross-link. This low affinity contact of alpha 83-92 and the receptor is sufficient for receptor activation and is crucial for the full understanding of the mechanistics of the receptor activation steps.

Lutropin, a pituitary hormone, and human choriogonadotropin bind to the same receptors in the ovary and elicit identical responses. A photoactivable derivative of human choriogonadotropin was used to identify the lutropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycylglycine, and iodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself did but with a slightly lower Ka, 2.98 X 10(9) M-1 compared with 5.1 X 10(9) M-1 for 125I-hormone. The binding could be blocked with untreated hormone or lutropin but not with follitropin, prolactin, insulin, or bovine serum albumin. Its alpha and beta subunits could be crosslinked to produce alpha beta dimer by photolysis, the extent of crosslinking being dependent upon the reagent concentration used for the derivatization: 22.8% at 50 microM, 37.3% at 100 microM, and 67.2% at 150 microM. When the 125I-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels under reducing conditions, three new bands of lower electrophoretic mobility appeared in addition to alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis and the presence of both cells bearing the receptor and the 125I-hormone derivative. It could be blocked by excess untreated hormone. The three bands correspond to molecular weights, 96,000 +/- 6,700, 66,000 +/- 4,600, and 63,000 +/- 4,400. Because the hormone has a high carbohydrate content and such glycoproteins are known to exhibit anomalous electrophoretic mobilities, these estimates must be tentative.

The luteinizing hormone/choriogonadotropin receptor, a seven-transmembrane receptor, is composed of two equal halves, the N-terminal extracellular exodomain and the C-terminal membrane-associated endodomain. Unlike most seven-transmembrane receptors, the exodomain alone is responsible for high affinity hormone binding, whereas signal is generated in the endodomain. These physical separations of hormone-binding and receptor activation sites are attributed to unique mechanisms for hormone binding and receptor activation of this receptor and its subfamily members. However, the precise hormone contact sites in the exodomain are unclear. In the preceding article (Hong, S., Phang, T., Ji, I., and Ji, T. H. (1998) J. Biol. Chem. 273, 13835-13840), a region immediately downstream of the N terminus of the exodomain was shown to be crucial for hormone binding. To test if the region interacts with the hormone, human choriogonadotropin (hCG) was photoaffinity-labeled with a peptide mimic corresponding to Gly18-Tyr36 of the receptor. This peptide mimic specifically photoaffinity-labeled both the alpha- and beta-subunits of hCG. Interestingly, hCGalpha was preferentially labeled. On the other hand, denatured hCG was not labeled, and a mutant analog of the peptide failed to label hCG. Furthermore, the affinity labeling was UV-dependent and saturable, indicating the specificity of the photoaffinity labeling. Our results indicate that the region of the exodomain interacts with hCG and that the contact points are near both subunits of hCG. Particularly, the alternate residues (Leu20, Cys22, and Gly24) are crucial for hCG binding. In addition, the results underscore the fact that there is a crucial hormone contact site outside of the popularly believed primary hormone-binding site that is composed of Leu-rich repeats and is located in the middle of the exodomain. Our observations are crucial for understanding the molecular mechanism through which the initial high affinity hormone binding leads to receptor activation in the endodomain.

The luteinizing hormone/choriogonadotropin receptor is a seven-transmembrane receptor. Unlike most seven-transmembrane receptors, it is composed of two halves of equal size, the N-terminal extracellular exodomain and the C-terminal membrane-associated endodomain. The exodomain is exclusively responsible for high affinity hormone binding, whereas receptor activation occurs only in the endodomain. This mutually exclusive physical separation of the two functional domains sets the lutropin receptor and its subfamily of receptors apart from all other seven-transmembrane receptors. The mechanisms of hormone binding and receptor activation also appear to be different from those of other receptors in that binding occurs in at least two steps. However, the precise hormone contact sites in the exodomain are unknown. To determine the hormone/receptor contact sites, we have examined the receptor using progressive truncation from the C terminus, Ala scanning, immunofluorescence microscopy, and antibody binding. Progressive truncation from the C terminus of the receptor indicates several discrete regions that impact hormone binding. These regions are around the boundaries of exons 1-2, 4-5, 6-7, and 9-10. Ala scanning of the Asp17-Arg26 region near the exon 1-2 junction uncovered three alternating residues (Leu20, Cys22, and Gly24) crucial for hormone binding. Ala substitution for any one of these residues abolished hormone binding, although the resulting mutant receptors were successfully expressed on the cell surface. In contrast, Ala substitution for their flanking and intervening residues did not impair hormone binding. These results and the data in the accompanying article (Phang, T., Kundu, G., Hong, S., Ji, I., and Ji, T. (1998) J. Biol. Chem. 273, 13841-13847) indicate that this region directly contacts the hormone and suggest a novel mode of embracing the hormone.

The region between the 10th and 12th cysteine (Cys88-Cys105 in human thyroid-stimulating hormone beta-subunit (hTSHbeta)) of the glycoprotein hormone beta-subunits corresponds to the disulfide-linked seat-belt region. It wraps around the common alpha-subunit and has been implicated in regulating specificity between human choriogonadotropin (hCG) and human follicle-stimulating hormone (hFSH), but determinants of hTSH specificity are unknown. To characterize the role of this region for hTSH, we constructed hTSH chimeras in which the entire seat-belt region Cys88-Cys105 or individual intercysteine segments Cys88-Cys95 and Cys95-Cys105 were replaced with the corresponding sequences of hCG and hFSH or alanine cassettes. Alanine cassette mutagenesis of hTSH showed that the Cys95-Cys105 segment of the seat-belt was more important for TSH receptor binding and signal transduction than the Cys88-Cys95 determinant loop region. Replacing the entire seat-belt of hTSHbeta with the hCG sequence conferred full hCG receptor binding and activation to the hTSH chimera, whereas TSH receptor binding and activation were abolished. Conversely, introduction of the hTSHbeta seat-belt sequence into hCGbeta generated an hCG chimera that bound to and activated the TSH receptor but not the CG/lutropin (LH) receptor. In contrast, an hTSH chimera bearing hFSH seat-belt residues did not possess any follitropic activity, and its thyrotropic activity was only slightly reduced. This may in part be due to the fact that the net charge of the seat-belt is similar in hTSH and hFSH but different from hCG. However, exchanging other regions of charge heterogeneity between hTSHbeta and hFSHbeta did not confer follitropic activity to hTSH. Thus, exchanging the seat-belt region between hTSH and hCG switches hormonal specificity in a mutually exclusive fashion. In contrast, the seat-belt appears not to discriminate between the TSH and the FSH receptors, indicating for the first time that domains outside the seat-belt region contribute to glycoprotein hormone specificity.

Affinity cross-linking of the lutropin receptor with 125I-human choriogonadotropin (hCG) on porcine granulosa cells produced four distinct homone-receptor complexes under reducing conditions. They contain 18-, 24-, 28-, and 34-kDa components (Ji, I., Bock, J. H., and Ji, T. H. (1985) J. Biol. Chem. 260, 12815-12821). Photoaffinity labeling and cross-linking produced 136-, 102-, and 74-kDa hCG-receptor complexes under reducing conditions and the 136-kDa complex under nonreducing conditions. In addition, the unreduced 102-kDa complex was seen in photoaffinity labeling but not in cross-linking. When the unreduced 136-kDa complex was reduced, the 102- and 74-kDa complexes were generated, indicating release of the 34- and the 28-kDa components in two steps. When the unreduced 102-kDa complex was reduced, the 74-kDa complex was produced, indicating the release of a 28-kDa component. The 74-kDa complex could not be reduced but was cleaved by alkaline treatment to produce the hCG alpha beta dimer. The results indicate that the 24-kDa component is released from the 74-kDa complex, since the apparent mass of the hCG alpha beta dimer on gels is 50 kDa. The 24-kDa component appears to be the initial site for photoaffinity labeling or cross-linking and to be disulfide linked to the 28-kDa component which is in turn disulfide linked to the 34-kDa component. These intercomponent disulfides exist in some receptors but not all. Formation of the disulfide-linked 136-kDa band required the presence of a sulfhydryl-blocking agent, N-ethylmaleimide. In particular, the 34-kDa component was vulnerable to reduction. There was no significant evidence of disulfides between the hormone and any of the receptor components.

A photoactivatable derivative of human follitropin was used to identify the follitropin receptor on porcine granulosa cells. The hormone was condensed with a heterobifunctional reagent, the N-hydroxysuccinimide ester of 4-azidobenzoylglycine, and radioiodinated. The 125I-labeled hormone (125I-hormone) derivative associated with the same number of receptors as 125I-hormone itself, but with a slightly lower Ka, 1.12 X 10(10) M-1 compared with 1.4 X 10(10) M-1 for the 125I-hormone. The binding could be blocked with untreated hormone. Its alpha and beta subunits could be cross-linked to produce alpha beta dimer by photolysis. When the 125I-hormone derivative bound to the cells was photolyzed for crosslinking and the products resolved by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, two new bands (106 and 61 kDa) of lower electrophoretic mobility appeared in addition to the alpha, beta, and alpha beta bands. Formation of these crosslinked complexes required photolysis, and the 125I-hormone derivative specifically bound to cells bearing the receptor. Binding could be blocked by excess untreated follitropin but not with human choriogonadotropin and thyrotropin. Under nonreducing conditions, one major band (104 kDa) of cross-linked complexes appeared. Upon reduction with dithiothreitol and second-dimensional electrophoresis, the 104-kDa band produced two smaller complexes of 75 and 61 kDa, indicating the loss of two components and the existence of intercomponent disulfides. Successful production of the 104-kDa complex requires blocking of free sulfhydryl groups with N-ethylmaleimide. It is, however, independent of various protease inhibitors or the temperature and the time period of hormone incubation with cells or the plasma membrane fraction. The mass estimates and the interaction with the hormone of the photoaffinity-labeled components are discussed.

The lutropin/choriogonadotropin receptor is a seven-transmembrane receptor and consists of two major domains of similar size, an extracellular exodomain and a membrane-associated endodomain which includes 3 exoloops. The uniquely large exodomain is responsible for high affinity hormone binding whereas receptor activation occurs at the endodomain. However, little is known about the relationship between the exodomain and endodomain. It was reported that hormone binding to the exodomain was improved when the endodomain was truncated. This result suggests that hormone binding to the exodomain was influenced by the endodomain. To test this hypothesis, amino acids of exoloop 2 were examined by Ala substitutions. The binding affinity was enhanced by some Ala substitutions but attenuated by others. These results indicate that exoloop 2 influences the hormone binding to the exodomain. Particularly, the high affinity hormone binding at the exodomain is constrained by a group of amino acids, Ser484, Asn485, Lys488, Ser490, and Ser499. Computer modeling suggests these residues may be positioned on one side of exoloop 2. It also influences the affinity for cAMP induction and the maximal cAMP production in distinct ways, in addition to its influence on the hormone binding affinity. The distinct ways of influencing these functions are sometimes in conflict and compromised to attain the maximal affinity for cAMP induction. As a result, the exodomain attains the maximal affinity for hormone binding when the endodomain is truncated and cAMP induction is disengaged.

The lutropin/choriogonadotropin receptor is a seven-helix transmembrane (TM) receptor. A unique feature of TM helices is the content of Pro, which generally is absent in alpha helices of globular proteins. Because Pro disrupts helices and introduces a approximately 26 degrees kink, it has been speculated that Pro plays a crucial role in the structure of TM helices, exoloops, and cytoloops of TM receptors. To examine the roles of the five TM Pros of the lutropin/choriogonadotropin receptor, these residues were individually substituted. Mutant receptors were examined for surface expression, hormone binding, and cAMP induction. Surface expression was monitored after introducing the flag epitope into the receptors. Flag epitopes slightly affected cAMP induction but not hormone binding or surface expression of receptors as monitored by immunofluorescence microscopy and 125I-anti-flag antibody. The results indicate that Pro479 in TM 4 and Pro598 in TM 7 play important yet contrasting roles. Pro479 is crucial for hormone binding at the cell surface but not after solubilization of the receptor. This is more likely due to the Pro side chain than the Pro-induced kink. Pro598 is important for surface expression. The kinks of Pro463 of TM 4, Pro562 of TM 6, or Pro591 of TM 7 are not important because the substitution of Phe for these residues did not significantly impact surface expression, hormone binding, and cAMP induction.

The luteinizing hormone/choriogonadotropin (CG) receptor belongs to a subfamily of glycoprotein hormone receptors within the seven-transmembrane receptor family. It is comprised of an extracellular N-terminal half of 341 amino acids and a membrane-associated C-terminal half of 303 amino acids. The N-terminal half is capable of high affinity hormone binding whereas the C-terminal half is capable of low affinity hormone binding and receptor activation. However, the precise location of the receptor activation site is currently unknown. We present evidence for the first time that Lys583 of exoloop 3 is crucial and irreplaceable for receptor activation to induce cAMP synthesis. Exoloop 3 is comprised of 11 amino acids and flanked by two Lys residues, Lys573 and Lys583, that are located at the boundaries with the transmembrane columns 6 and 7, respectively. All substitutions including Arg for Lys583 did not affect the high affinity human CG binding, but they resulted in the complete loss of cAMP synthesis induced by human CG. Ala substitutions of the other amino acids in exoloop 3 did not make such a dramatic impact on cAMP induction. The Ala scan revealed two distinct groups of amino acids in terms of their importance in cAMP induction, one group being more important than the other. Interestingly, these two groups of amino acids are arranged in an alternate sequence. This result suggests a specific structure similar to a beta-like structure for exoloop 3.

The follicle-stimulating hormone (FSH) receptor is a member of the glycoprotein hormone receptor subfamily of the seven-transmembrane receptor superfamily. These receptors have an extracellular N-terminal half of approximately 350 amino acids and a membrane-associated C-terminal half of approximately 350 amino acids. The N-terminal halves have the high affinity hormone binding site. On the other hand, the C-terminal half of the lutropin/choriogonadotropin receptor has the receptor activation site. However, little is known about the activation site and mechanism of the FSH receptor, although the existing evidence indicates crucial differences in the activation of the FSH receptor and the lutropin/choriogonadotropin receptor. As a first step to resolve this issue, we examined the upstream juxtamembrane five amino acids, Asp405-Ile406-His407-Thr408-Lys409, of the exoloop 1. Ala scan and multi-substitutions show that the five amino acid sequence is important for both hormone binding and receptor activation to induce cAMP synthesis, despite its short length. Specifically, His407 is important for high affinity hormone binding, whereas Asp405, Thr408, and Lys409 are crucial for receptor activation. The data suggest that the five amino acids may form a turn of helix that is an extension of the transmembrane helix 2. In this helical arrangement, Asp405, Thr408, and Lys409 are grouped to form a hydrophilic face of the helix, suggesting a correlation between this arrangement and receptor activation. In addition, the diverse and differential roles of the five amino acids indicate that high affinity hormone binding and receptor activation are discernible functions. These novel observations will be helpful for understanding the activation mechanism of the FSH receptor.

Environmentally friendly toxins of Bacillus thuringiensis are effective in controlling agriculturally and biomedically harmful insects. However, little is known about the insect receptor molecules that bind these toxins and the mechanism of insecticidal activity. We report here for the first time the cloning and expression of a cDNA that encodes a receptor (BT-R1) of the tobacco hornworm Manduca sexta for an insecticidal toxin of B. thuringiensis. The receptor is a 210-kDa membrane glycoprotein that specifically binds the cryIA(b) toxin of B. thuringiensis subsp. berliner and leads to death of the hornworm. BT-R1 shares sequence similarity with the cadherin superfamily of proteins.

Human choriogonadotropin (hCG) is a heterodimeric hormone composed of an alpha and a beta subunit. hCG and its asialo (ashCG) and deglycosylated (dghCG) forms vary in their ability to stimulate hormone responsive adenylate cyclase. ashCG is a partial agonist, and dghCG is an antagonist. Photoactivatable moieties were coupled to hCG, ashCG, and dghCG, and the derivatives were radioiodinated. Competitive binding studies indicate that all of the derivatives had a similar affinity for the gonadotropin receptor on porcine granulosa cell membranes. Radiolabeled derivatives were used to photoaffinity label the gonadotropin receptor. Radiolabeled complexes were separated by NaDodSO4/PAGE. All of the derivatives produced similar autoradiographic patterns, except that dghCG produced an additional 48-kDa complex. To investigate the structure of the complexes further, peptide mapping of proteolytic digests was used. All, except for the 48-kDa complex, generated similar peptide maps indicating a relationship between those complexes in which the smaller components are part of the larger. The 48-kDa complex contained both subunits of 40-kDa dghCG. Therefore, this complex is expected to contain an additional component of 8 kDa. The complex was generated whether the hormone-receptor complex was photoaffinity labeled on cells, on isolated membranes, or after solubilizing in detergent. Formation was blocked by excess hCG and did not occur in the absence of UV irradiation. We conclude that the hCG derivatives are able to photoaffinity label the hCG receptor but that the dghCG derivative can photoaffinity label an additional component that was not observed when derivatives of hCG or ashCG were used to label the receptor.

The electrophoretic mobility of radioiodinated follitropin (FSH) alpha and beta subunits as well as the alpha beta dimer changed markedly depending on the concentration of reducing agents such as dithiothreitol. The changes were more dramatic in the beta subunit than in the alpha subunit. 125I-FSH, complexed to the receptor on porcine granulosa cells or in Triton X-100 extracts, was cross-linked with a cleavable (nondisulfide) homobifunctional reagent, solubilized in sodium dodecyl sulfate without reducing agents, and electrophoresed. The cross-linked sample revealed three bands of high molecular mass, in addition to the hormone subunit and dimer bands. The band of lightest mass, 110 kDa, was the major band and the other two of 76 and 62 kDa were barely noticeable. Upon reduction with dithiothreitol, the 110-kDa band decreased while the 76- and 62-kDa bands increased, indicating the existence of disulfides between components of the 110-kDa complex. Formation of the disulfide-linked complexes requires 125I-FSH, specifically bound to the hormone receptor and cross-linking, and can be prevented with an excess of native FSH but not human choriogonadotropin. Complex formation was independent of blocking free sulfhydryl groups with N-ethylmaleimide. When the cross-linked complexes were reduced in the gel matrix and analyzed on fresh gels, the 76- and 62-kDa complexes were generated from the 110-kDa band, indicating the loss of two components. The lost components were estimated to be at 14 and 34 kDa. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22-, 14-, and 34-kDa components to the FSH alpha beta dimer. The results of reduction of the cross-linked complexes demonstrate the existence of disulfide linkage between the three components.

Both of the alpha and beta subunits of intact human follitropin (FSH) were radioiodinated with 125I-sodium iodide and chloramine-T and could be resolved on sodium dodecyl sulfate-polyacrylamide gels. Radioiodinated FSH was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to its membrane receptor on the porcine granulosa cell surface as well as to a Triton X-100-solubilized form of the receptor. Cross-linked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65, 83, and 117 kDa, in addition to the hormone bands. The hormone alpha beta dimer band corresponded to 43 kDa. Formation of the three bands requires the 125I-hormone to bind specifically to the receptor with subsequent cross-linking. Binding was prevented by an excess of the native hormone but not by other hormones. A monofunctional analog of the cross-linking reagent failed to produce the three bands. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of cross-linked complexes were treated to cleave covalent cross-links and then electrophoresed in a second dimension, 18-, 22-, and 34-kDa components were released, in addition to the alpha and beta subunits of the hormone.

Radioiodinated human choriogonadotropin was affinity-cross-linked with a cleavable (nondisulfide) homobifunctional reagent to the hormone receptor on porcine granulosa cells and the solubilized sample was electrophoresed. Cross-linked samples revealed four additional bands of slower electrophoretic mobility in addition to the hormone alpha, beta, and alpha beta dimer bands. The four bands corresponded to masses of 68, 74, 102, and 136 kDa whereas the alpha beta dimer band corresponded to 50 kDa. Formation of the four bands requires the 125I-hormone to bind specifically to the receptor with subsequent cross-linking. Binding can be prevented by excess of native hormone but not by follitropin. A monofunctional analog of the cross-linking reagent failed to produce the four bands. They were also produced by cross-linking Triton X-100-solubilized hormone-receptor complexes. Reagent concentration-dependent cross-linking revealed that their formation was sequential; smaller complexes formed first and then larger ones. When gels of the cross-linked sample were treated with reagents that cleave covalent cross-links and then electrophoresed in a second dimension gel, 18-, 24-, 28-, and 34-kDa components were released, in addition to the alpha and beta subunits of the native hormone. Simultaneous peptide mapping of the cross-linked complexes in the gel matrix with Staphylococcus V8 protease or papain revealed progressive proteolysis to generate terminal fragments of 30 or 27 kDa, respectively. These fragments were unique to and commonly present in the 74-, 102-, and 136-kDa hormone-receptor complexes but were not produced by proteolysis of the cross-linked human choriogonadotropin (hCG) alpha beta dimer or the hCG alpha subunit. Apparently, the radioactively labeled segment(s) of the alpha subunit of 125I-hCG was cross-linked to the 24-kDa component. The results demonstrate the protein nature of the receptor and suggest that 125I-hCG was initially cross-linked to the 24-kDa component to generate the 74-kDa complex, then the 28- and 34-kDa components were sequentially cross-linked to the 24-kDa component in the 74-kDa complex to generate the 102- and 134-kDa complexes.

It has been shown that a photoactivable derivative of human choriogonadotropin (hCG) labels the lutropin receptor on porcine granulosa cells [Ji, I. & Ji, T. H. (1980) Proc. Natl. Acad. Sci. USA 77, 7167-7170]. In an attempt to identify which of the hCG subunits labeled the receptor, three sets of different hCG derivatives were prepared. In the first set, hCG was coupled to the N-hydroxysuccinimide ester of 4-azidobenzoylglycine and radioiodinated. In the second set, only one of the subunits was radioiodinated, but both subunits were allowed to react with the reagent. In the third set, both the reagent and [125I]iodine were coupled to only one of the subunits. The binding activity of each hormone derivative was comparable to that of 125I-labeled hCG. After binding of these hormone derivatives to the granulosa cell surface, they were photolyzed. After solubilization, autoradiographs of sodium dodecyl sulfate/polyacrylamide gels of each sample revealed a number of labeled bands; the hCG derivatives containing 125I-labeled alpha subunit produced four bands (molecular weights 120,000 +/- 6,000, 96,000 +/- 5,000, 76,000 +/- 4,000, and 73,000 +/- 4,000) and those containing 125I-labeled beta subunit produced three bands (molecular weights 106,000 +/- 6,000, 88,000 +/- 5,000, and 83,000 +/- 4,000). Results were the same when the hormone-receptor complexes were solubilized in 0.5% Triton X-100 and then photolyzed or when the hormone was derivatized with a family of reagents having arms of various lengths. We conclude that both the alpha subunit and the beta subunit of hCG photoaffinity labeled certain membrane polypeptides and that these polypeptides are related to the hormone receptor.

The human follicle-stimulating hormone receptor (FSH-R) consists of two distinct domains of >330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain. The exodomain alone binds hormone with high affinity, whereas the endodomain is the site of receptor activation. Coordination of these two domains is essential for successful hormone action but little is known about their functional and structural relationship. In this communication, we report that exoloop 3 of FSH-R constrains follicle-stimulating hormone binding to the exodomain. When the FSH-R exodomain was prepared by truncating its endodomain, the hormone binding affinity of the exodomain was slightly improved, compared with the wild type receptor. The binding affinity was further improved by >3-fold when the exodomain was attached to the membrane-associated domain of CD8. These results suggest that the FSH-R endodomain attenuates hormone binding at the exodomain. As a first step to test this hypothesis, the 11 amino acids except Ala589 of exoloop 3 were individually substituted with Ala. Ala substitution for Leu583 or Ile584 improved the hormone binding affinity by 4-6-fold while totally abolishing cAMP induction, indicating an inverse relationship. The Ala substitution for Lys580 or Pro582 had a similar trend but to a lesser extent. This significant improvement in the binding affinity suggests that the four residues at the N-terminal region of exoloop 3 interact with the exodomain and constrain the hormone binding in the wild type receptor. This effect is specific since substitutions for other than the 4 residues did not improve the hormone binding affinity. Computer modeling shows that the 4 residues can be positioned on one side of exoloop 3. This result and the apparent inverse relationship of hormone binding and cAMP induction suggest that these two essential functions may work against each other. Therefore, hormone binding might be compromised to preserve cAMP inducibility while maintaining a reasonably high, but below maximum, binding affinity.

One pot hydrothermal method is used for synthesis of groundnuts-like samarium oxide (Sm2O3) thin film on stainless steel substrate. The Sm2O3film is characterized by X-ray diffraction, water contact angle, UV–visible spectrophotometer, photoluminescence, and field emission scanning electron microscopy techniques. The hydrothermal method allows the formation of cubic Sm2O3film with porous groundnuts-like morphology. The Sm2O3film is hydrophilic with the optical band gap of 3.70 eV. Electrochemical capacitive behavior of Sm2O3film is studied using cyclic voltammetry, galvanostatic charge–discharge measurement and electrochemical impedance spectroscopy. The Sm2O3film exhibits maximum specific capacitance of 155 Fg−1at 5 mVs−1scan rate in 1 M KOH electrolyte.