Structural organization of the nine spectrin repeats of Kalirin.

Sequence analysis suggests that KALRN, a Rho GDP/GTP exchange factor genetically linked to schizophrenia, could contain as many as nine tandem spectrin repeats (SRs). We expressed and purified fragments of Kalirin containing from one to five putative SRs to determine whether they formed nested structures that could endow Kalirin with the flexible rodlike properties characteristic of spectrin and dystrophin. Far-UV circular dichroism studies indicated that Kalirin contains nine SRs. On the basis of thermal denaturation, sensitivity to chemical denaturants, and the solubility of pairs of repeats, the nine SRs of Kalirin form nested structures. Modeling studies confirmed this conclusion and identified an exposed loop in SR5; consistent with the modeling, this loop was extremely labile to proteolytic cleavage. Analysis of a direpeat fragment (SR4:5) encompassing the region of Kalirin known to interact with NOS2, DISC-1, PAM, and Arf6 identified this as the least stable region. Analytical ultracentrifugation indicated that SR1:3, SR4:6, and SR7:9 were monomers and adopted an extended conformation. Gel filtration suggested that ΔKal7, a natural isoform that includes SR5:9, was monomeric and was not more extended than SR5:9. Similarly, the nine SRs of Kal7, which was also monomeric, were not more extended than SR5:9. The rigidity and flexibility of the nine SRs of Kal7, which separate its essential N-terminal Sec14p domain from its catalytic domain, play an essential role in its contribution to the formation and function of dendritic spines.

Investigation of the structural requirements for peptide precursor processing in AtT-20 cells using site-directed mutagenesis of proadrenocorticotropin/endorphin.

Mouse pro-ACTH/endorphin (or POMC) contains in its sequence each of the four possible pairs of basic amino acids recognized as potential cleavage sites in the production of bioactive peptides from higher mol wt precursors: KR (lysine-arginine), RR, RK, and KK. To examine the structural requirements for processing and routing in one region of pro-ACTH/endorphin, a reporter mutation was introduced into the mouse pro-ACTH/endorphin cDNA; a methionine residue was mutated to an isoleucine residue to allow biosynthetic double labeling with [3H]Ile and [35S]Met. Analysis of stable cell lines expressing the reporter cDNA indicated that this mutation did not affect processing or secretion. Therefore, additional mutations were introduced on the reporter background to investigate important structural features of the precursor. First, the tripeptide signal for N-linked glycosylation in the N-terminal glycopeptide (Asn65,Ser66,Ser67) was disrupted by the conservative substitution of asparagine65 with a glutamine residue. Secondly, O-glycosylation was prevented by substitution of threonine45 with an alanine residue. Finally, lysine50 was mutated to an arginine residue, transforming the RK doublet preceding the gamma 3MSH sequence into an RR doublet. The results show that the enzymatic machinery of AtT-20 cells fails to cleave efficiently at the Arg-Lys (RK) site even after elimination of any possible structural hindrance by carbohydrate side-chains. Elimination of O-linked oligosaccharides to the N-terminal side of gamma 3MSH did not allow cleavage at the RK site, and elimination of N-linked oligosaccharides did not alter the processing and routing of pro-ACTH/endorphin in AtT-20 cells. However, mutation of the RK sequence to RR allowed extensive cleavage regardless of the occurrence of O- or N-glycosylation.

Further characterization of peptidylglycine alpha-amidating monooxygenase from bovine neurointermediate pituitary.

The ability of purified bovine neurointermediate pituitary peptidyl glycine alpha-amidating monooxygenase to catalyze the conversion of peptide substrates (D-Tyr-X-Gly) into amidated product peptides (D-Tyr-X-NH2) was evaluated. The pH optimum of the reaction was pH 8.5 when X was Val, Trp, or Pro, but 5.5 to 6.0 when X was Glu. Similar maximum velocity (Vmax) values were obtained for the Val, Trp, and Pro substrates while the Glu substrate had a substantially higher Vmax. The Michaelis-Menten constant (Km) of the enzyme for the peptide substrate increased in the order Trp less than Val less than Pro much less than Glu. Increasing levels of ascorbate brought about parallel increases in Km and Vmax, suggesting the presence of an irreversible step separating the interaction of the enzyme with the two substrates. The effect of copper on enzyme activity was dependent on the peptide substrate and the reaction pH. With the Val substrate, exogenous copper was required for optimal activity; no other metal ion tested could substitute for copper. With the Glu substrate, exogenous copper was not required for optimal activity; however, diethyldithiocarbamate, a copper chelator, inhibited activity and only copper could reverse this inhibitory effect. The ability of various cofactors to stimulate alpha-amidating monooxygenase activity was also dependent on assay conditions. With the Val or Glu substrate in the presence of exogenous copper, a variety of cofactors in addition to ascorbate were capable of supporting activity. With the Glu substrate in the absence of exogenous copper, the requirement of the enzyme for ascorbate was more strict. In keeping with the proposed reaction mechanism, nearly 1 mol ascorbate was consumed for each mole of D-Tyr-Glu-NH2 produced.

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.

Structure of a receptor-binding fragment from human luteinizing hormone beta-subunit determined by [1H]- and [15N]nuclear magnetic resonance spectroscopy.

The structure of the glycoprotein hormones (LH, CG, FSH, and TSH) and their mechanism of receptor recognition are problems of long-standing interest and speculation. Here we describe the two-dimensional [1H]nuclear magnetic resonance ([1H]NMR) analysis of a linear peptide model for the intercysteine sequence (38-57) from the beta-subunit of human (h) LH. This sequence contains functional determinants for receptor binding and postreceptor activation and is predicted by computer-based modeling to fold as a compact minidomain containing a central amphipathic helix. To test this prediction, an Arg-extended disulfide-free (38-57) analog of enhanced solubility was prepared for complementary circular-dichroic and two-dimensional NMR studies. The linear peptide retains ovarian membrane receptor-binding activity. Although the peptide is not highly structured in aqueous solution, circular-dichroic analysis shows partial alpha-helix formation in a lipophilic medium (50% trifluoroethanol). Complete sequential assignment is obtained in 50% trifluoroethanol based on homonuclear and [15N]edited heteronuclear NMR methods. alpha-Helix-related (i,i + 3) connectivities are observed by nuclear-Overhauser effect spectroscopy that define an amphipathic alpha-helical segment (residues 41-48). Additional long range nuclear-Overhauser effects are observed in the C-terminal region that are consistent with beta-turns involving one or more proline residues; these may serve to reverse the direction of the peptide chain. A nuclear-Overhauser effect contact is identified between residues 38 and 55 at opposite ends of the linear sequence, suggesting that a loop configuration is significantly populated in this solvent system. These results, taken together, characterize elements of ordered structure in the 38-57 peptide, which appear to be distinguishing features of hLH (and the homologous region of hCG). We propose that the structure of this peptide provides a model for the structure of the corresponding region of native hLH in the hormone-receptor complex.

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.

The NH2-terminal proregion of peptidylglycine alpha-amidating monooxygenase facilitates the secretion of soluble proteins.

A highly conserved ten amino acid proregion separates the peptidylglycine alpha-hydroxylating monooxygenase (PHM) domain of the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) protein from the NH2-terminal signal peptide; propeptides with amino acid sequences similar to the PAM proregion have been identified in other secreted proteins. In AtT-20 cells, but not in human embryonic kidney (hEK)-293 cells, an endogenous endoprotease acting at a site distal to the trans-Golgi network efficiently removes the propeptide from stably transfected monofunctional PHM (PHMs). We constructed a mutant PHM protein (delta ProPHMs) in which the proregion was deleted and the signal peptide joined directly to the monooxygenase domain. Newly synthesized, enzymatically active delta ProPHMs was secreted from both AtT-20 cells and hEK-293 cells more slowly than PHMs. In endocrine cells, the proregion was not required for storage in regulated secretory granules. We transferred the PAM proregion to prohormone convertase 2 (PC2), another soluble constituent of secretory granules, to determine whether the effect of the proregion were transferrable. In both AtT-20 cells and hEK-293 cells, the PAM/PC2 fusion molecule was able to exit the endoplasmic reticulum more rapidly than PC2.

Structure of the precursor to an enzyme mediating COOH-terminal amidation in peptide biosynthesis.

Many bioactive peptides terminate with an amino acid alpha-amide at their COOH terminus. The enzyme responsible for this essential posttranslational modification is known as peptidyl-glycine alpha-amidating monooxygenase or PAM. We identified cDNAs encoding the enzyme by using antibodies to screen a bovine intermediate pituitary lambda gt11 expression library. Antibodies to a beta-galactosidase/PAM fusion protein removed PAM activity from bovine pituitary homogenates. The 108,207 dalton protein predicted by the complete cDNA is approximately twice the size of purified PAM. An NH2-terminal signal sequence and short propeptide precede the NH2 terminus of purified PAM. The sequences of several PAM cyanogen bromide peptides were localized in the NH2-terminal half of the predicted protein. The cDNA encodes an additional 430 amino acid intragranular domain followed by a putative membrane spanning domain and a hydrophilic cytoplasmic domain. The forms of PAM purified from bovine neurointermediate pituitary may be generated by endoproteolytic cleavage at a subset of the 10 pairs of basic amino acids in the precursor. High levels of PAM mRNA were found in bovine pituitary and cerebral cortex. In corticotropic tumor cells, levels of PAM mRNA and pro-ACTH/endorphin mRNA were regulated in parallel by glucocorticoids and CRF.

Interaction of nascent preproparathyroid hormone molecules with microsomal membranes.

To characterize the early steps in the interaction of nascent chains of preproparathyroid hormone (prepro-PTH) with the secretory apparatus, such truncated nascent chains still attached to ribosomes were tested for binding to microsomal membranes and cleavage by signal peptidase. Nascent chains of 114, 97, 88, 81, 70, and 59 residues were tested for their ability to bind tightly to membranes and to undergo signal sequence cleavage. Chains of 81 residues and longer bound tightly to the membranes and were cleaved by signal peptidase. The 88- and 81-residue precursors and their corresponding pro-proteins were less efficiently associated with the membranes than were the 114- and 97-residue precursors and their corresponding pro-proteins. The 70-residue chain bound to the membrane but was not cleaved. When this peptide was subsequently released from the ribosome with puromycin, it was cleaved by signal peptidase. The 59-residue chain bound only slightly to the microsomal membrane and was not cleaved by signal peptidase, even when the nascent peptide was released from the ribosome with puromycin. Thus the critical length for productive binding to microsomal membranes is between 59 and 70 residues; the length required for signal cleavage is between 70 and 81 residues.

A subunit interaction site in human luteinizing hormone: identification by photoaffinity cross-linking.

The sites of noncovalent association between the alpha- (common) and beta- (hormone-specific) subunits in the glycoprotein hormones [LH, human CG (hCG), FSH, and TSH] remain to be completely defined. This information is essential to efforts to map the three-dimensional structure of the hormones and to help explain the stability of the hormone heterodimer in the circulation. Among numerous approaches that have been employed to identify these sites of subunit interaction, chemical or photoaffinity cross-linking has the advantage of identifying the complementary sites of contact on the respective subunits. We have used the stable photoaffinity ligand, L-benzoylphenylalanine (Bpa), to evaluate subunit interaction by the hLH beta sequence (1-15). A peptide from this region of hCG beta has been shown previously to inhibit subunit association. The 3H-labeled Bpa was incorporated into the peptide at position 8 (Trp) during solid-phase synthesis. After incubation with alpha under long-wavelength (366 nm) ultraviolet light, a Bpa- (1-15)-labeled-alpha-fraction was isolated. Control experiments showed the binding to be covalent and specific to (1-15). Two other Bpa-labeled beta-fragments, the receptor-binding loop (38-57) and the (30-43) peptide containing the highly conserved CAGY sequence, did not cross-link. The site of contact in alpha was localized by peptide mapping and sequence analysis to the N-terminal fragment (18-33), most likely at Met-29 or Gly-30. Both the alpha- and beta-sites are adjacent to or overlapping receptor-binding regions. Subunit contact sites and receptor-binding segments may thus be oriented in close proximity to provide a multicomponent receptor-binding domain imparting full activity to the native hormone.

Carbohydrate modifications transform human chorionic gonadotropin into a potent stimulator of adenosine 3',5'-monophosphate and growth responses in FRTL-5 thyroid cells.

To delineate the role of carbohydrate moiety in the expression of in vitro thyrotropic activity of hCG, its variants that lacked sialic acid residues or the entire carbohydrate moiety on one or both subunits were prepared. They along with intact hCG were then tested for the abilities to bind to TSH receptor and stimulate cAMP production and growth responses in FRTL-5 cells. The removal of sialic acid from either one or both subunits sharply increased the [125I]bovine TSH binding-inhibiting activity of hCG in the receptor assay. Among the variants tested, desialylated hCG (as-hCG) was the most potent inhibitor, followed by alpha-as-beta, as-alpha-beta, and hCG in that order. With respect to their abilities to stimulate cAMP generation in the cells, the activities of all desialylated hCG variants were markedly higher than that of hCG itself, and as in the receptor assay, as-hCG was the most potent stimulator tested. At the same concentration (100 micrograms/ml), as-alpha-beta, alpha-as-beta, and hCG were approximately only 57%, 46%, and 27% as active as as-hCG in activating cAMP production. The findings in the growth assay were entirely consistent with those noted in the cAMP response assay. Among the deglycosylated hybrids examined, alpha-dg-beta was the most effective in activating cAMP release. An equivalent dose (200 micrograms/ml) was about 1.7, 2.7, and 3.8 times as active as dg-hCG dg-alpha-beta, and hCG, respectively. The deglycosylated variants of hCG showed a similar pattern of activity in growth response and cAMP accumulation assays. In both cases, alpha-dg-beta was the most potent stimulator, while hCG was the least active. No significant difference between the potencies of dg-alpha-beta and alpha-dg-beta was discerned in the receptor assay; however, deglycosylated hCG (dg-hCG) was sharply more active. Results of these studies strongly suggest that the optimal expression of in vitro thyrotropic activity of hCG variants in FRTL-5 cells may require an alpha-subunit, with intact carbohydrate, in combination with the deglycosylated beta-subunit. Further, they demonstrate that modification of the hCG carbohydrate moiety alone can transform it from a weak agonist to a potent stimulator of in vitro thyrotropic bioactivity. These results also provide further evidence to support the notion that the degree of expression of thyrotropic activity is strongly affected by the species in which the studies are performed.

A functional determinant in human luteinizing hormone and chorionic gonadotropin: differential effect of mutations about beta-GLN-54.

There is evidence that the conserved glutamine at residue 54 in the beta-subunit of human LH and and CG (hCG) is important for biological activity. Mutation to Arg in LH has been reported to impair receptor binding, leading to a documented case of hypogonadism, whereas in hCG the mutation has been shown to result in defective subunit association. Functional distinctions between LH and hCG have been described, but the significance of peptide-chain differences between the two has not been investigated systematically. We therefore compared the role of Gln-54 and its neighboring residues in both hormones, through replacement by amino acids with contrasting properties using site-directed mutagenesis. The mutant subunits were coexpressed with alpha-subunit in mammalian (Chinese hamster ovary) cells and the secreted hormones assayed for heterodimer formation, receptor binding, and steroidogenesis in murine Leydig cell tumor (MA-10) cells. Basic (Arg, Lys) substitution for Gln-54 in either hormone markedly impaired subunit association (<20% of wild-type) and the heterodimers that were formed were inactive (<5% of wild-type) in both assays. Arg-substituted hCG was also inactive in an adenylate cyclase assay using HEK-293 cells expressing rat LH/hCG receptor. After acidic (Glu) or neutral (Ala) substitution, heterodimer formation was less impaired (50-60% of wild-type), but effects on receptor interaction differed between the two hormones. The LH mutants still lacked binding activity, whereas the hCG products were fully active. The importance of residue 54 for receptor interaction appears to be sharply localized because mutation at adjacent positions (Pro-53 and Val-55) did not impair the activity of either hormone. Diminished heterodimer formation by Ile-53 mutation in LH (but not hCG), together with the similar effects of basic mutations at 54, imply long-distance effects as these residues are remote from alpha in the crystal structure. Our findings indicate that position 54 in LH and hCG is a determinant for both subunit association and receptor interaction. The differing responses between LH and hCG to certain mutations suggest that structural characteristics of the peptide chains may confer functional differences despite their close sequence homology.

Characterization and application of a radioimmunoassay for reduced, carboxymethylated human luteinizing hormone alpha-subunit.

We have established a double antibody RIA using a rabbit antiserum prepared against reduced, carboxymethylated (RCXM) human LH alpha-subunit, with RCXM-alpha as tracer and standard. This antiserum did not cross-react with any native gonadotropins or subunit, and reacted only weakly with RCXM-alpha. A tryptic digest of RCXM alpha-subunit was completely reactive, while chymotryptic digestion abolished all immunoreactivity. By testing with separate tryptic fragments, the recognition site could be localized to a segment close to the amino-terminus of the peptide chain. When applied to measurement of serum and urine, an immunoreactive species, parallel to RCXM alpha-subunit by serial dilution, was found in concentrations of 1-2 ng/ml in serum and 3-4 ng/ml in urine. Similar levels of the immunoreactive component were found in conditions of elevated gonadotropins (e.g. pregnancy) as well as gonadotropin deficiency(panhypopituitarism and Kallmann's syndrome). After stimulation with LHRH, no rise was noted at times up to 6 h despite the fact that both LH and LH-alpha were elevated. The data indicate that the sequence-specific antiserum may be detecting an immunoreactive form of alpha-subunit of LH whose kinetics of appearance and disappearance differs from those of the native subunit.

Studies on the isolation and chemical-physical properties of porcine follicle-stimulating hormone.

Methods are described for isolating highly purified FSH from porcine pituitary glands. The biological activity of pure material was 22 NIH-FSH-P2 U/mg, as judged by the ovarian weight augmentation bioassay. Virtually no immunoreactive LH was indicated by RIA, and only one component was detected by both gel electrophoresis and immunoprecipitation. Amino acid and carbohydrate analyses of the highly purified FSH indicated the presence of 1-tryptophan, a high content of aspartic and glutamic acids, and 6% sialic acid. Molecular weights of untreated and guanidine-treated porcine FSH were estimated from hydrodynamic measurements of Stokes radii and sedimentation coefficients. A highly specific and sensitive RIA was also developed for this hormone.

Analysis of human follistatin structure: identification of two discontinuous N-terminal sequences coding for activin A binding and structural consequences of activin binding to native proteins.

A primary physiological function of follistatin is the binding and neutralization of activin, a transforming growth factor-beta family growth factor, and loss of function mutations are lethal. Despite the critical biological importance of follistatin's neutralization of activin, the structural basis of activin's binding to follistatin is poorly understood. The purposes of these studies were 1) to identify the primary sequence(s) within the N-terminal domain of the follistatin coding for activin binding, and 2) to determine whether activin binding to the native protein causes changes in other structural domains of follistatin. Synthetic peptide mimotopes identified within a 63-residue N-terminal domain two discontinuous sequences capable of binding labeled activin A. The first is located in a region (amino acids 3-26) of follistatin, a site previously identified by directed mutagenesis as important for activin binding. The second epitope, predicted to be located between amino acids 46 and 59, is newly identified. Although the sequences 3-26 and 46-59 code for activin binding, native follistatin only binds activin if disulfide bonding is intact. Furthermore, pyridylethylation of Cys residues followed by N-terminal sequencing and amino acid analysis revealed that all of the Cys residues in follistatin are involved in disulfide bonds and lack reactive free sulfhydryl groups. Specific ligands were used to probe the structural effects of activin binding on the other domains of the full-length molecule, comprised largely of the three 10-Cys follistatin module domains. No effects on ligand binding to follistatin-like module I or II were observed after the binding of activin A to native protein. In contrast, activin binding diminished recognition of domain III and enhanced that of the C domain by their respective monoclonal antibody probes, indicating an alteration of the antigenic structures of these regions. Thus, subsequent to activin binding, interactions are likely to occur between regions of follistatin located in different domains and separated by considerable lengths of linear sequence. Such interactions could have important functional significance with respect to the structural heterogeneity of naturally occurring follistatins.

Structure activity relationship of parathyroid hormone: separation of the hypotensive and the hypercalcemic properties.

The present study was conducted to show that the hypercalcemic and the vascular relaxing activities of PTH are two separable properties. During the hypotensive action of the synthetic fragment bovine (b) PTH-(1-34), plasma calcium levels were not significantly changed. Mild oxidation with hydrogen peroxide abolished the hypotensive and hypercalcemic actions of bPTH-(1-84). However, the same treatment on bPTH-(1-34) abolished only the hypotensive and not the hypercalcemic action. Analysis of the amino acid composition revealed only the oxidation of the methionines to methionine sulfoxides. The other amino acids remained unchanged. In addition, the analog with methionines replaced by norleucine, [Nle8,Nle18,Tyr34]bPTH-(1-34), was active in all the vascular assays, and these activities were unaffected by hydrogen peroxide treatment of the molecule. Perhaps the methionine sulfoxides in the hydrogen peroxide-treated bPTH-(1-34) affected the changes of the molecule in such a manner that the part of the molecule for the vascular action but not that for the hypercalcemic action was no longer accessible to the receptors of the target organs. The hypotensive pentapeptide, bPTH-(24-28), was not active in the hypercalcemic assay. All these data are consistent with our hypothesis that the vascular relaxing and the hypercalcemic actions of PTH are two separate properties of the molecule.

The carbohydrate moiety of bovine thyrotropin is essential for full bioactivity but not for receptor recognition.

TSH is a glycoprotein hormone whose carbohydrate content varies among different species. Although recent studies suggest that variants of TSH deficient in carbohydrate occur naturally, the significance of the carbohydrate moiety of TSH in respect to its thyrotropic function is unclear. The present studies were undertaken, therefore, to examine this question. A highly purified preparation of bovine TSH (bTSH) was deglycosylated by treatment with anhydrous hydrogen fluoride. Amino acid and carbohydrate analyses of the original and deglycosylated preparations indicated that approximately 85% of the carbohydrate originally present had been removed and that the protein moiety was unaltered. As judged from TSH radioreceptor assays, bTSH and deglycosylated bTSH (dg-bTSH) bound to human thyroid membranes with equal affinity, since both caused a half-maximal inhibition of [125I]bTSH binding at approximately equal concentrations. Nonetheless, dg-bTSH at optimal concentration displayed only about one third the activity of intact TSH in stimulating adenylate cyclase activity in human thyroid membranes. dg-bTSH also antagonized the adenylate cyclase-stimulating activity of intact bTSH in this system, but only weakly, since abolition of the bTSH effect required an approximately 40-fold higher concentration of dg-bTSH. In cultures of FRTL5 cells, a cloned line of follicular cells derived from normal rat thyroid, both intact and dg-bTSH enhanced cell growth, as measured by [3H]thymidine incorporation and stimulated cAMP release in the medium, but the response elicited by dg-bTSH was much less than that caused by equal concentrations of the intact hormone. In accord with the findings in the in vitro assays, dg-bTSH evoked a much smaller response than bTSH did in the in vivo mouse assay. It is concluded that although not required for receptor recognition, the carbohydrate moiety of bTSH is essential for the full expression of its biological activity.

Rat parathyroid hormone-(1-34) fragment: renal adenylate cyclase activity and receptor binding properties in vitro.

The rat PTH molecule contains five sequence differences from either the bovine or the human hormone within the biologically active 1-34 region. A synthetic rat 1-34 peptide was tested for activity by in vitro activation of canine and rat renal adenylate cyclase and binding to canine renal membrane receptors. The mean potency of 21,400 Medical Research Council units/mg in the canine adenylate cyclase system and 24,900 in the rat system was 8- to 10-fold higher than human 1-34 and 2- to 4-fold greater than bovine 1-34. These values represent the highest potency we have observed to date for a PTH preparation by these assay systems. In contrast, receptor binding of the rat fragment was comparable to that of bovine and human 1-34. Half-maximal inhibition of radioligand binding occurred at 1.7- 2.0 X 10(-9) M with all synthetic hormones. Hence, the amino acid substitutions in rat 1-34 appear to affect the cyclase-activating sequence domain without increasing avidity for the receptor. Analogs combining the rat sequence with modifications known to enhance receptor binding and/or retard enzymatic degradation offer a promising approach to the preparation of still more potent parathyroid agonists.

Analysis of parathyroid hormone's principal receptor-binding region by site-directed mutagenesis and analog design.

Previous deletion studies established that the 25-34 region of PTH is important for receptor binding. We used oligonucleotide-directed mutagenesis to generate 47 different mutations in this region of human (h) PTH-(1-84) and evaluated cAMP-stimulating activity in ROS 17/2.8 cells. The hydrophobic residues Leu24 and Leu28 stood out as mutationally intolerant sites, while neighboring polar residues were comparatively tolerant. A series of synthetic PTH analogs was designed to test these residues further. The affinity of [Tyr34]hPTH-(1-34)NH2 for ROS 17/2.8 cells [dissociation constant (Kd), approximately 5 nM)] was dramatically reduced by the substitution of either Leu24 or Leu28 with Glu (Kd, approximately 20,000 and 8,000 nM, respectively). The Val31-->Glu substitution also sharply reduced affinity (Kd, approximately 200 nM). In contrast, the nearby charge-reversing change of Asp30-->Lys had no effect on binding affinity (Kd, approximately 5 nM). Similar effects were observed in the opposum kidney cell line. The binding of [Tyr34]hPTH-(15-34)NH2 to ROS 17/2.8 and opposum kidney cells (Kd, approximately 10 microM) was abolished by Glu substitutions at position 24, 28, or 31; the Lys30 change was without effect. These results suggest that the adverse effects of the Glu substitutions on receptor binding are not due purely to the disruption of tertiary interactions with the 1-14 region. Circular dichroism spectroscopy indicated that the substitutions do not affect local helical structure. The data suggest that Leu24, Leu28, and Val31 contribute important receptor-binding interactions and are consistent with the hypothesis that an amphipathic alpha-helix in the carboxy-terminal region of PTH-(1-34) is involved in receptor binding.

Determinants of [Arg2]PTH-(1-34) binding and signaling in the transmembrane region of the parathyroid hormone receptor.

Previously, we reported that [Arg2]PTH-(1-34) bound to the rat osteosarcoma cell line, ROS 17/2.8, with 2-fold higher apparent affinity than it did to the opossum kidney cell line, OK, yet the analog was only a weak partial agonist for cAMP stimulation with ROS 17/2.8 cells, whereas it was a full cAMP agonist with OK cells. These results suggested that the rat and opossum PTH receptors differ in a region recognized by the hormone's amino-terminus. In this report we show that the cloned PTH receptors derived from ROS 17/2.8 and OK cells, expressed in COS-7 cells, also displayed altered responses to [Arg2]PTH-(1-34). Thus, [Arg2]PTH-(1-34) bound to the cloned rat PTH receptor with 7-fold higher affinity than it did to the cloned opossum PTH receptor, and in cAMP stimulation assays, it was a much weaker agonist with the rat receptor than it was with the opossum receptor. Studies with rat/opossum PTH receptor chimeras suggested that the membrane-spanning region of the receptor contributed to the different binding and signaling responses to [Arg2]PTH-(1-34). Point mutation analysis identified three sites in or near the extracellular ends of transmembrane domains V and VI, which specifically affected [Arg2]PTH-(1-34) binding and signaling.