Disruption of the first extracellular loop of thyrotropin receptor prevents ligand binding

In order to understand the function of the first extracellular loop of the human thyrotropin receptor (hTSHR), each of two peptides of nine amino acids was inserted into the first extracellular loop of hTSHR. hTSHR cDNA was subcloned into the eukaryotic expression vector, pRc/CMV (hTSHR/pRc/CMV). B-hTSHR/pRc/CMV, a mutant hTSHR cDNA which encodes a hydrophilic peptide insert (AGTTRRVAI) and C-hTSHR/pRc/CMV which encodes a hydrophobic peptide insert (ATVLVVPMI) between +486 Ileu and +487 Asp of hTSHR were transfected into Chinese hamster ovary cells to generate the B-1 and C-6 cell lines, respectively. Neither thyrotropin (TSH) nor thyroid stimulating antibody (TSAb) stimulated cAMP production by B-1 or C-6 cells. An 125I-TSH binding assay showed that neither cell line bound TSH. Our data demonstrated that these mutations impaired both TSH binding and cAMP production. This evidence suggests that the first extracellular loop of hTSHR may have a crucial role in the TSH- and TSAb-dependent signal transduction.     

Deletions in the third intracellular loop of the thyrotropin receptor. A new mechanism for constitutive activation

Gain-of-function mutations of the thyrotropin receptor (TSHR) gene have been invoked as one of the major causes of toxic thyroid adenomas. In a toxic thyroid nodule, we recently identified a 9-amino acid deletion (amino acid positions 613-621) within the third intracellular (i3) loop of the TSHR resulting in constitutive receptor activity. This finding exemplifies a new mechanism of TSHR activation and raises new questions concerning the function of the i3 loop. Because the i3 loop is thought to be critical for receptor/G protein interaction in many receptors, we systematically reexamined the role of the TSHR's i3 loop for G protein coupling. Thus, various deletion mutants were generated and functionally characterized. We identified an optimal deletion length responsible for constitutive activity. If the number of deleted amino acids was reduced, elevated basal cAMP accumulation was found to be concomitantly diminished. Expansion of the deletion dramatically impaired cell surface expression of the receptor. Shifting the deletion toward the N terminus of the i3 loop resulted in unaltered strong constitutive receptor activity. In contrast, translocation of the deletion toward the C terminus led to significantly reduced basal cAMP formation, most probably due to destruction of a conserved cluster of amino acids. In this study, we show for the first time that amino acid deletions within the i3 loop of a G protein-coupled receptor result in constitutive receptor activity. In the TSHR, 75% of the i3 loop generally assumed to play an essential role in G protein coupling can be deleted without rendering the mutant receptor unresponsive to thyrotropin. These findings support a novel model explaining the molecular events accompanying receptor activation by agonist.     

Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation

FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine erythropoietin receptor cDNA survive and proliferate in the presence of erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the erythropoietin receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the erythropoietin receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the erythropoietin receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by erythropoietin was decreased in cells expressing erythropoietin receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP-1 cells.     

Tyrosine 474 of ZAP-70 is required for association with the Shc adaptor and for T-cell antigen receptor-dependent gene activation

The protein tyrosine kinase ZAP-70 plays a central role in T-cell activation. Following receptor engagement, ZAP-70 is recruited to the phosphorylated subunits of the T-cell antigen receptor (TCR). This event results in ZAP-70 activation and in association of ZAP-70 with a number of signaling proteins. Among these is the Shc adaptor, which couples the activated TCR to Ras. Shc interaction with ZAP-70 is mediated by the Shc PTB domain. The inhibitory effect of a Shc mutant containing the isolated PTB domain suggests that Shc interaction with ZAP-70 might be required for TCR signaling. Here, we show that a point mutation (Phe474) of the putative Shc binding site on ZAP-70, spanning tyrosine 474, prevented ZAP-70 interaction with Shc and the subsequent binding of Shc to phospho-zeta. Neither ZAP-70 catalytic activity nor the pattern of protein phosphorylation induced by TCR triggering was affected by this mutation. However expression of the Phe474 ZAP-70 mutant resulted in impaired TCR-dependent gene activation. ZAP-70 could effectively phosphorylate Shc in vitro. Only the CH domain, which contains the two Grb2 binding sites on Shc, was phosphorylated by ZAP-70. Both Grb2 binding sites were excellent substrates for ZAP-70. The data show that Tyr474 on ZAP-70 is required for TCR signaling and suggest that Shc association with ZAP-70 and the resulting phosphorylation of Shc might be an obligatory step in linking the activated TCR to the Ras pathway.     

Glycine-receptor activation is required for receptor clustering in spinal neurons

The ability of nerve cells to receive up to several thousands of synaptic inputs from other neurons provides the anatomical basis for information processing in the vertebrate brain. The formation of functional synapses involves selective clustering of neurotransmitter receptors at presumptive postsynaptic regions of the neuronal plasma membrane. Receptor-associated proteins are believed to be crucial for this process. In spinal neurons, synaptic targeting of the inhibitory glycine receptor (GlyR) depends on the expression of the anchoring protein gephyrin. Here we show that the competitive GlyR antagonist strychnine and L-type Ca2+-channel blockers inhibit the accumulation of GlyR and gephyrin at postsynaptic membrane areas in cultured rat spinal neurons. Our data are consistent with a model in which GlyR activation that results in Ca2+ influx is required for the clustering of gephyrin and GlyR at developing postsynaptic sites. Similar activity-driven mechanisms may be of general importance in synaptogenesis.     

Kennedy's disease: caspase cleavage of the androgen receptor is a crucial event in cytotoxicity

X-linked spinal and bulbar muscular atrophy (SBMA), Kennedy's disease, is a degenerative disease of the motor neurons that is associated with an increase in the number of CAG repeats encoding a polyglutamine stretch within the androgen receptor (AR). Recent work has demonstrated that the gene products associated with open reading frame triplet repeat expansions may be substrates for the cysteine protease cell death executioners, the caspases. However, the role that caspase cleavage plays in the cytotoxicity associated with expression of the disease-associated alleles is unknown. Here, we report the first conclusive evidence that caspase cleavage is a critical step in cytotoxicity; the expression of the AR with an expanded polyglutamine stretch enhances its ability to induce apoptosis when compared with the normal AR. The AR is cleaved by a caspase-3 subfamily protease at Asp146, and this cleavage is increased during apoptosis. Cleavage of the AR at Asp146 is critical for the induction of apoptosis by AR, as mutation of the cleavage site blocks the ability of the AR to induce cell death. Further, mutation of the caspase cleavage site at Asp146 blocks the ability of the SBMA AR to form perinuclear aggregates. These studies define a fundamental role for caspase cleavage in the induction of neural cell death by proteins displaying expanded polyglutamine tracts, and therefore suggest a strategy that may be useful to treat neurodegenerative diseases associated with polyglutamine repeat expansions.     

Understanding the thyrotropin receptor function-structure relationship

The thyrotropin (TSH) receptor (TSHR) is a key protein in the control of thyroid function and a major thyroid autoantigen. Recently, molecular cloning of the receptor has been carried out and we now review the impact of this work on our understanding of the physiology and pathophysiology of the TSHR. Analysis of recombinant TSHR proteins expressed in prokaryotic and eukaryotic systems has indicated that post-translational processing is important for the formation of active receptors. Studies of TSHR glycosylation have shown that a 'mature' form of the receptor containing mainly complex-type sugar residues is principally involved in TSH and TSHR autoantibody (TRAb) binding. In addition, the processing of the TSHR peptide chain into two subunits observed with native TSHR has been confirmed using recombinant TSHR. However, despite considerable efforts in many laboratories, the binding site(s) for TSH and TRAb on the TSHR have not been well characterized as yet and lessons learned from the discovery of naturally occurring amino acid mutations of the TSHR confirm the complexity of the hormone and autoantibody binding sites. Future progress in producing large amounts of pure TSHR as well as monoclonal TRAbs, followed by crystallographic analysis of TSHR-TSH complexes and TSHR-TRAb complexes, should be helpful in providing a better insight into the relationship between TSHR structure and function.     

The extracellular thyrotropin receptor domain is not a major candidate for mutations in toxic thyroid nodules

Constitutive activation of the cyclic adenosine monophosphate (cAMP) cascade by either thyrotropin receptor (TSHR) or gsp mutations is considered to be the major molecular cause of toxic thyroid nodules (TTNs). In a recent study we investigated a consecutive series of 31 TTNs and identified 15 somatic TSHR mutations (n = 14 in exon 10; n = 1 in exon 9) but no mutations in gsp exons 7-10. The purpose of the present study was to determine whether the extracellular TSHR domain would be a candidate for mutations causing TTNs. Therefore, we screened TSHR exons 1-8 in the remaining 16 TTNs without mutations in TSHR exons 9 and 10 and gsp exons 7-10 of our previous study. Except for a known functional polymorphism in exon 1 (Pro 52 Thr) in 2 TTNs and a silent base exchange in exon 7 (187 Asn) in 7 other TTNs no TSHR mutations were identified. To clarify the molecular etiology of TTNs without TSHR or gsp mutations, candidate genes in other steps of the cAMP cascade have to be considered.     

Mediation of cyclic AMP signaling by the first intracellular loop of the gonadotropin-releasing hormone receptor

The gonadotropin-releasing hormone (GnRH) receptor, which is a unique G protein-coupled receptor without a C-terminal cytoplasmic domain, activates both inositol phosphate (InsP) and cAMP signaling responses. The function of the highly basic first intracellular (1i) loop of the GnRH receptor in signal transduction was evaluated by mutating selected residues located in its N and C termini. Replacements of Leu58, Lys59, Gln61, and Lys62 at the N terminus, and Leu73, Ser74, and Leu80 at the C terminus, caused no change in binding affinity. The agonist-induced InsP and cAMP responses of the Q61E and K59Q,K62Q receptors were also unaffected, but the L58A receptor showed a normal InsP response and an 80% decrease in cAMP production. At the C terminus, the InsP response of the L73R receptor was normal, but cAMP production was reduced by 80%. The EC50 for GnRH-induced InsP responses of the S74E and L80A receptors was increased by about one order of magnitude, and the cAMP responses were essentially abolished. These findings indicate that cAMP signaling from the GnRH receptor is dependent on specific residues in the 1i loop that are not essential for activation of the phosphoinositide signaling pathway.     

NMDA receptor activation during status epilepticus is required for the development of epilepsy

NMDA receptor activation has been implicated in modulating seizure activity; however, its complete role in the development of epilepsy is unknown. The pilocarpine model of limbic epilepsy involves inducing status epilepticus (SE) with the subsequent development of spontaneous recurrent seizures (SRSs) and is widely accepted as a model of limbic epilepsy in humans. The pilocarpine model of epilepsy provides a tool for looking at the molecular signals triggered by SE that are responsible for the development of epilepsy. In this study, we wanted to examine the role of NMDA receptor activation on the development of epilepsy using the pilocarpine model. Pretreatment with the NMDA receptor antagonist MK-801 does not block the onset of SE in the pilocarpine model. Thus, we could compare animals that experience similar lengths of SE in the presence or absence of NMDA receptor activation. Animals treated with MK-801 (4 mg/kg) 20 min prior to pilocarpine (350 mg/kg) (MK-Pilo) were compared to the pilocarpine treated epileptic animals 3-8 weeks after the initial episode of SE. The pilocarpine-treated animals displayed both ictal activity and interictal spikes on EEG analysis, whereas MK-801-pilocarpine and control animals only exhibited normal background EEG patterns. In addition, MK-801-pilocarpine animals did not exhibit any SRSs, while pilocarpine-treated animals exhibited 4.8 +/- 1 seizures per 40 h. MK-801-pilocarpine animals did not demonstrate any decrease in pyramidal cell number in the CA1 subfield of the hippocampus, while pilocarpine animals averaged 15% decrease in cell number. In summary, the MK-801-pilocarpine animals exhibited a number of characteristics similar to control animals and were statistically significantly different from pilocarpine-treated animals. Thus, NMDA receptor inhibition by MK-801 prevented the development of epilepsy and interictal activity following SE. These results indicate that NMDA receptor activation is required for epileptogenesis following SE in this model of limbic epilepsy.     

An antagonist peptide-EPO receptor complex suggests that receptor dimerization is not sufficient for activation

Dimerization of the erythropoietin (EPO) receptor (EPOR), in the presence of either natural (EPO) or synthetic (EPO-mimetic peptides, EMPs) ligands is the principal extracellular event that leads to receptor activation. The crystal structure of the extracellular domain of EPOR bound to an inactive (antagonist) peptide at 2.7 A resolution has unexpectedly revealed that dimerization still occurs, but the orientation between receptor molecules is altered relative to active (agonist) peptide complexes. Comparison of the biological properties of agonist and antagonist EMPs with EPO suggests that the extracellular domain orientation is tightly coupled to the cytoplasmic signaling events and, hence, provides valuable new insights into the design of synthetic ligands for EPOR and other cytokine receptors.     

Aspartate-407 in Rhodobacter sphaeroides cytochrome c oxidase is not required for proton pumping or manganese binding

Several pathways for proton transport in cytochrome c oxidase have been proposed on the basis of mutational analysis and X-ray structure: at least one for moving "pumped" protons from the interior to exterior of the membrane and a separate route for transporting "substrate" protons from the interior to the binuclear metal center to combine with oxygen to make H2O. According to the crystal structures of cytochrome c oxidase, Asp407 (Rhodobacter sphaeroides numbering) is at the interface of subunit I and subunit II of the oxidase, in a negative patch proposed to be the proton exit site in a pumping pathway, as well as a possible ligand to Mg [Iwata et al. (1995) Nature 376, 660-669]. Three mutants at the Asp407 position of R. sphaeroides cytochrome oxidase, Asp407Ala, Asp407Asn, and Asp407Cys, have been purified and characterized. All showed electron transfer activity, and pH dependence of activity, similar to that of the wild type enzyme and no major structural changes, as evidenced by visible, EPR, and resonance Raman spectroscopy. When reconstituted into artificial vesicles, the purified mutants pumped protons with normal efficiency and responded to the membrane pH and electrical gradients in a manner similar to that of wild type. Furthermore, the EPR spectra and Mn quantitation analysis of mutants grown in high Mn indicated no significant alteration in the Mn/Mg site. These results suggest that Asp407 does not play a critical role in proton translocation or in Mn/Mg binding.     

A 76-amino acid disulfide loop in the Yersinia pseudotuberculosis invasin protein is required for integrin receptor recognition

The Yersinia pseudotuberculosis invasin protein is a 986-amino acid protein that promotes bacterial penetration into mammalian cells by avidly binding multiple beta 1-chain integrins. A 192-amino acid carboxyl-terminal domain of invasin was previously shown to be sufficient for binding. Evidence is presented here that a 76-amino acid disulfide loop in the integrin binding domain of invasin is required for invasin-mediated cell binding and entry. Bacterial mutants that were altered at either of 2 cysteine residues in the binding domain of invasin were completely defective for entry. Purified invasin protein derivatives altered at either of these cysteines, in contrast to the wild-type invasin, did not promote either cell binding or penetration. Analysis of proteolytic products of invasin in the presence or absence of reducing agent provided evidence of an intra-chain disulfide bond near the carboxyl terminus of the protein. Alkylation of invasin derivatives with [3H]iodoacetate indicated that these 2 cysteines were normally disulfide-bonded. A treatment that resulted in the maximal reduction of the disulfide bond also resulted in maximal loss of cell attachment activity. These results indicate that the 76-amino acid disulfide loop at the carboxyl terminus of invasin is required for recognition by integrins.     

Mutations in the activation loop tyrosine of the oncoprotein v-Fps

Mutations were made in the activation loop tyrosine of the kinase domain of the oncoprotein v-Fps to assess the role of autophosphorylation in catalysis. Three mutant proteins, Y1073E, Y1073Q, and Y1073F, were expressed and purified as fusion proteins of glutathione-S-transferase from Escherichia coli and their catalytic properties were evaluated. Y1073E, Y1073Q, and Y1073F have k(cat) values that are reduced by 5-, 35-, and 40-fold relative to the wild-type enzyme, respectively. For all mutant enzymes, the Km values for ATP and a peptide substrate, EAEIYEAIE, are changed by 0.4-2-fold compared to the wild-type enzyme. The slopes for the plots of relative turnover versus solvent viscosity [(k(cat))eta] are 0.71 +/- 0.08, 0.10 +/- 0.06, and approximately 0 for wild type, Y1073Q, and Y1073E, respectively. These results imply that the phosphoryl transfer rate constant is reduced by 19- and 130-fold for Y1073E and Y1073Q compared to the wild-type enzyme. The dissociation constant of the substrate peptide is 1.5-2.5-fold lower for the mutants compared to wild type. The inhibition constant for EAEIFEAIE, a competitive inhibitor, is unaffected for Y1073E and raised 3-fold for Y1073Q compared to wild type. Y1073E and Y1073Q are strongly activated by free magnesium to the same extent and the apparent affinity constant for the metal is similar to that for the wild-type enzyme. The data indicate that the major role of autophosphorylation in the tyrosine kinase domain of v-Fps is to increase the rate of phosphoryl transfer without greatly affecting active-site accessibility or the local environment of the activating metal. Finally, the similar rate enhancements for phosphoryl transfer in v-Fps compared to protein kinase A [Adams et al. (1995) Biochemistry 34, 2447-2454] upon autophosphorylation suggest a conserved mechanism for communication between the activation loop and the catalytic residues of these two enzymes.     

Expression of recombinant human thyrotropin receptor in myeloma cells

Starting with a previously isolated cDNA for human thyrotropin receptor (TSHR), we established a transformed myeloma cell line, SP56, which expresses human TSHR on its cell surface. Binding analysis showed that SP56 bears 1.1 x 10(5) TSHR per cell with a Kd of 2.2 x 10(-10) M. Using the purified cellular membrane, we established a TSH binding inhibition immunoglobulin (TBII) assay for autoantibodies against TSHR. We compared it with the TBII assay utilizing porcine thyroid membranes expressing porcine TSHR, which has been widely used for TBII assay, by using 96 serum samples from patients with autoimmune thyroid disease and normal individuals. Our TBII assay was more sensitive than the one using porcine TSHR: of 38 sera of patients which were judged negative for autoantibodies to TSHR (TBII value below 10%) by the latter assay, 28 were positive (above 20%) in our assay. By using a perfusion culture system, we obtained as many as 3 x 10(10) SP56 cells, from which 3,450 mg protein of the membrane could be purified; this is sufficient for 15,000 assays. The results indicate that the membrane of the myeloma cell line SP56 is more suitable for use in the TBII assay than the porcine thyroid membrane, in terms of sensitivity to autoantibodies against TSHR in human sera.     

Arginine 206 of the C5a receptor is critical for ligand recognition and receptor activation by C-terminal hexapeptide analogs

C5a is a 74-amino-acid glycoprotein whose receptor is a member of the rhodopsin superfamily. While antagonists have been generated to many of these receptors, similar efforts directed at family members whose natural ligands are proteins have met with little success. The recent development of hexapeptide analogs of C5a has allowed us to begin elucidation of the molecular events that lead to activation by combining a structure/activity study of the ligand with receptor mutagenesis. Removal of the hexapeptide's C-terminal arginine reduces affinity by 100-fold and eliminates the ability of the ligand to activate the receptor. Both the guanidino side chain and the free carboxyl of the arginine participate in the interaction. The guanidino group makes the energy-yielding contact with the receptor, while the free carboxylate negates "electrostatic" interference with Arg-206 of the receptor. It is the apparent movement Arg-206 induced by this set of interactions that is responsible for activation, since conversion of Arg-206 to alanine eliminates the agonist activity of the hexapeptides. Surprisingly, activation is a nearly energy-neutral event and may reflect the binding process rather than the final resting site of the ligand.     

Point mutations of the thyrotropin receptor determining structural requirements for its ability to bind thyrotropin and to stimulate adenylate cyclase activity

The two cysteines C494 and C569, located in the first and second extracellular loop, respectively, of the thyrotropin (TSH) receptor, were mutated to serines to test the functional significance of the putative disulfide bond between these two cysteines. Single (C494S and C569S) and double (C494/569S) mutant receptors were generated, transiently expressed in COS cells, and compared with regard to the ability to bind ligand and to mediate stimulation of adenylate cyclase activity. The double mutant retained ligand binding capacity, in contrast to the single cysteine mutants that were essentially devoid of binding capacity. The ability of the mutated receptor variants to stimulate adenylate cyclase activity was lost or greatly reduced.     

Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas

The pituitary hormone thyrotropin stimulates the function, expression of differentiation and growth of thyrocytes by cyclic AMP-dependent mechanisms. Tissue hyperplasia and hyperthyroidism are therefore expected to result when activation of the adenylyl cyclase-cAMP cascade is unregulated. This is observed in several situations, including when somatic mutations impair the GTPase activity of the G protein Gsa (ref 6, 7). Such a mechanism is probably responsible for the development of a minority of monoclonal hyperfunctioning thyroid adenomas. Here we identify somatic mutations in the carboxy-terminal portion of the third cytoplasmic loop of the thyrotropin receptor in three out of eleven hyperfunctioning thyroid adenomas. These mutations are restricted to tumour tissue and involve two different residues (aspartic acid at position 619 to glycine in two cases, and alanine at position 623 to isoleucine in one case). The mutant receptors confer constitutive activation of adenylyl cyclase when tested by transfection in COS cells. This shows that G-protein-coupled receptors are susceptible to constitutive activation by spontaneous somatic mutations and may thus behave as proto-oncogenes.     

Constitutive activation of cyclic AMP but not phosphatidylinositol signaling caused by four mutations in the 6th transmembrane helix of the human thyrotropin receptor

Four different somatic mutations (F631C, T632I, D633E, and D633Y) in the putative 6th transmembrane helix of the human thyrotropin receptor (TSHR) were recently described in hyperfunctioning thyroid adenomas [Porcellini et al. (1994) J. Clin. Endocrinol. Metab. 79, 657-661]. We transiently expressed these mutant receptors in Cos-7 cells and measured [125I]TSH binding, basal and TSH-stimulated cAMP production, and phosphatidylinositol hydrolysis. The concentration of receptors expressed at the cell surface was lower for the mutants than for the wild type (WT) TSHR. Compared to the WT, all four mutant receptors caused a marked increase in basal cAMP levels, but did not increase basal production of inositol phosphates. This suggests that autonomous thyroid function and adenoma formation may be related to constitutive activation of the cAMP pathway alone. A cluster of conserved residues at the base of the 6th transmembrane helix of the TSHR and other glycoprotein hormone receptors appears important for maintaining an inactive receptor conformation.