Mutations in the mouse TSH receptor equivalent to human constitutively activating TSH receptor mutations also cause constitutive activity.

Constitutively activating mutations in the human thyroid-stimulating hormone (TSH) receptor (TSHr) have been identified as the most prevalent molecular cause of non-autoimmune hyperthyroidism. To investigate the feasibility of an animal model for non-autoimmune hyperthyroidism, we introduced two mutations in the mouse TSHr which had previously been identified in the human TSHr. The two human mutations showed strong differences in TSH binding, basal cAMP and IP accumulation. In the human TSHr, the Ile 486 Phe mutation causes a high increase of basal cAMP accumulation and also basal stimulation of the inositol phosphate pathway, whereas the Val 509 Ala mutation results in a low increase of basal cAMP accumulation without affecting IP signaling. RNA was isolated from mouse thyroid tissue and reverse transcribed. A 2.4 kb PCR product from the mouse TSHr was cloned into the pGEM-T vector system. Ile was substituted with Phe at codon 486 and Val with Ala at codon 509. These mutated mouse TSHrs were subcloned in the pSVL expression vector. After transient expression in COS-7 cells, basal and TSH-stimulated cAMP and IP accumulation, cell surface expression and TSH binding were determined and directly compared to the human TSHr. Whereas constitutively activating mutations of the human parathyroid hormone (PTH)/PTH-related peptide receptor showed little or no change in basal cAMP accumulation when introduced into the rat PTH/PTHrP receptor, these two mouse TSHr mutations resulted in constitutive activity similar to the homologous mutations in the human TSHr. Therefore, it should be possible to establish a mouse model for non-autoimmune hyperthyroidism by homologous recombination to study the pathogenetic mechanisms of non-autoimmune hyperthyroidism.     

A conserved Asn in transmembrane helix 7 is an on/off switch in the activation of the thyrotropin receptor

The thyrotropin (TSH) receptor is an interesting model to study G protein-coupled receptor activation as many point mutations can significantly increase its basal activity. Here, we identified a molecular interaction between Asp(633) in transmembrane helix 6 (TM6) and Asn(674) in TM7 of the TSHr that is crucial to maintain the inactive state through conformational constraint of the Asn. We show that these residues are perfectly conserved in the glycohormone receptor family, except in one case, where they are exchanged, suggesting a direct interaction. Molecular modeling of the TSHr, based on the high resolution structure of rhodopsin, strongly favors this hypothesis. Our approach combining site-directed mutagenesis with molecular modeling shows that mutations disrupting this interaction, like the D633A mutation in TM6, lead to high constitutive activation. The strongly activating N674D (TM7) mutation, which in our modeling breaks the TM6-TM7 link, is reverted to wild type-like behavior by an additional D633N mutation (TM6), which would restore this link. Moreover, we show that the Asn of TM7 (conserved in most G protein-coupled receptors) is mandatory for ligand-induced cAMP accumulation, suggesting an active role of this residue in activation. In the TSHr, the conformation of this Asn residue of TM7 would be constrained, in the inactive state, by its Asp partner in TM6.     

Constitutive activation of the thyroid-stimulating hormone receptor (TSHR) by mutating Ile691 in the cytoplasmic tail segment

Background

Autosomal dominant non-autoimmune hyperthyroidism (ADNAH) is a rare genetic disorder of the endocrine system. Molecular genetic studies in ADNAH have revealed heterozygous germline mutations in the TSHR. To data, mutations leading to an increase in the constitutive activation of the TSHR have been described in the transmembrane segments, exoloops and cytoplasmic loop of TSHR. These mutations result in constitutive activation of the G_αs/cAMP or G_αq/11/inositol phosphate (IP) pathways, which stimulate thyroid hormone production and thyroid proliferation.

Methodology/Principal Findings

In a previous study, we reported a new TSHR mutation located in the C-terminal domain of TSHR, which results in a substitution of the conserved Ile⁶⁹¹ for Phe. In this study, to address the question of whether the I691F mutated receptor could be responsible for G_αs/cAMP or G_αq/11/IP constitutive activity, wild-type and TSHR mutants were expressed in COS-7 cells to determine cAMP constitutive activity and IP formation. Compared to the cell surface with expression of the A623V mutated receptor as positive control, the I691F mutated receptor showed a slight increase of cAMP accumulation. Furthermore, I691F resulted in constitutive activation of the G_αq/11/IP signaling pathway.

Conclusions/Significance

Our results indicate that Ile⁶⁹¹ not only contributes to keeping TSHR inactive in the G_αs/cAMP pathways but also in the G_αq/11/IP cascade.     

Modulation of ligand selectivity associated with activation of the transmembrane region of the human follitropin receptor

Recently, three naturally occurring mutations in the serpentine region of the FSH receptor (FSHr) (D567N and T449I/A) have been identified in three families with spontaneous ovarian hyperstimulation syndrome (OHSS). All mutant receptors displayed abnormally high sensitivity to human chorionic gonadotropin and, in addition, D567N and T449A displayed concomitant increase in sensitivity to TSH and detectable constitutive activity. In the present study, we have used a combination of site-directed mutagenesis experiments and molecular modeling to explore the mechanisms responsible for the phenotype of the three OHSS FSHr mutants. Our results suggest that all mutations lead to weakening of interhelical locks between transmembrane helix (TM)-VI and TM-III, or TM-VI and TM-VII, which contributes to maintaining the receptor in the inactive state. They also indicate that broadening of the functional specificity of the mutant FSHr constructs is correlated to their increase in constitutive activity. This relation between basal activity and functional specificity is a characteristic of the FSHr, which is not shared by the other glycoprotein hormone receptors. It leads to the interesting suggestion that different pathways have been followed during primate evolution to avoid promiscuous stimulation of the TSHr and FSHr by human chorionic gonadotropin. In the hFSHr, specificity would be exerted both by the ectodomain and the serpentine portion.     

Role of cleavage and shedding in human thyrotropin receptor function and trafficking.

The thyrotropin receptor (TSHR) undergoes a cleavage at the cell membrane, leading to a heterodimer, comprising an alpha extracellular and a beta-transmembrane and intracellular subunits, held together by disulfide bonds. Moreover, part of the alpha-subunit of the receptor is shed from thyroid and transfected L cells. To understand the role of cleavage and shedding, we constructed deletion mutants starting, respectively, at the most N-terminal (S314), and C-terminal (L378) cleavage sites previously mapped, corresponding to free beta1 or beta2-subunits without further modification of receptor structure. Functional studies performed in COS-7 cells showed that both mutants display an increased basal activation of the cAMP pathway when compared with the wild-type receptor. By contrast, deletion of almost the entire extracellular domain of the receptor (TM409 mutant) totally impairs receptor function, thus confirming a role of the juxtamembrane extracellular region in receptor function. The beta1 mutant receptor exhibited an increased internalization when compared with the hormone-activated holoreceptor. Furthermore, no recycling was observed in the case of the beta1 mutant receptor. These observations strongly argue for a different conformation between the receptor activated by cleavage and shedding on the one hand, and the receptor activated by the ligand on the other hand. Cleavage and shedding of a receptor already activated by a transmembrane activating mutation M453T further increase its activity, showing that the extracellular domain still exerts a negative effect in the M453T holoreceptor. An increased internalization of the M453T receptor was observed when compared with the wild-type receptor, which was increased further in the corresponding truncated beta1-M453T receptor. Thus cleavage and shedding yield TSHR activation but also increase internalization of the free beta-subunits of the receptor, the latter mechanism limiting simultaneously excessive receptor signaling. The combined effects may be responsible for the limited basal constitutive activation of the cAMP pathway that is detected for the TSHR.     

An activated human follicle-stimulating hormone (FSH) receptor stimulates FSH-like activity in gonadotropin-deficient transgenic mice

FSH mediates its testicular actions via a specific Sertoli cell G protein-coupled receptor. We created a novel transgenic model to investigate a mutant human FSH receptor (FSHR(+)) containing a single amino acid substitution (Asp567Gly) equivalent to activating mutations in related glycoprotein hormone receptors. To examine the ligand-independent gonadal actions of FSHR(+), the rat androgen-binding protein gene promoter was used to direct FSHR(+) transgene expression to Sertoli cells of gonadotropin-deficient hypogonadal (hpg) mice. Both normal and hpg mouse testes expressed FSHR(+) mRNA. Testis weights of transgenic FSHR(+) hpg mice were increased approximately 2-fold relative to hpg controls (P < 0.02) and contained mature Sertoli cells and postmeiotic germ cells absent in controls, revealing FSHR(+)-initiated autonomous FSH-like testicular activity. Isolated transgenic Sertoli cells had significantly higher basal ( approximately 2-fold) and FSH-stimulated ( approximately 50%) cAMP levels compared with controls, demonstrating constitutive signaling and cell-surface expression of FSHR(+), respectively. Transgenic FSHR(+) also elevated testosterone production in hpg testes, in the absence of circulating LH (or FSH), and it was not expressed functionally on steroidogenic cells, suggesting a paracrine effect mediated by Sertoli cells. The FSHR(+) response was additive with a maximal testosterone dose on hpg testicular development, demonstrating FSHR(+) activity independent of androgen-specific actions. The FSHR(+) response was male specific as ovarian expression of FSHR(+) had no effect on hpg ovary size. These findings reveal transgenic FSHR(+) stimulated a constitutive FSH-like Sertoli cell response in gonadotropin-deficient testes, and pathways that induced LH-independent testicular steroidogenesis. This novel transgenic paradigm provides a unique approach to investigate the in vivo actions of mutated activating gonadotropin receptors.     

Comparative proteomic analysis to dissect differences in signal transduction in activating TSH receptor mutations in the thyroid

In the thyroid, cAMP controls both thyroid growth and function. Gain-of-function mutations in the thyroid-stimulating hormone receptor (TSHR) lead to constitutive cAMP formation and are a major cause of autonomous thyroid adenomas. The impact of activating TSHR mutations on the signal transduction network of the thyrocyte is not fully understood. To gain more insights into constitutive TSHR signaling, rat thyrocytes (FRTL-5 cells) with stable expression of three activating TSHR mutants (mutTSHR: A623I, L629F and Del613-621), which differ in their functional characteristics in vitro, were analyzed by a quantitative proteomic approach and compared to the wild-type TSHR (WT-TSHR). This study revealed (1) differences in the expression of Rab proteins suggesting an increased TSHR internalization in mutTSHR but not in the WT-TSHR; (2) differential stimulation of PI3K/Akt signaling in mutTSHR vs. WT-TSHR cells, (3) activation of Epac, impairing short-time Akt phosphorylation in both, mutTSHR and WT-TSHR cells. Based on the analysis of global changes in protein expression patterns, our findings underline the complexity of gain-of-function TSHR signaling in thyrocytes, which extends beyond pure cAMP and/or IP formation. Moreover, evidence for augmented endocytosis in the mutTSHR, adds to a new concept of TSHR signaling in thyroid autonomy. Further studies are required to clarify whether the observed differences in Rab, PI3K and Epac signaling may contribute to differences in the phenotypic presentation, i.e. stimulation of function and growth of thyroid autonomy in vivo.     

Activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to an agonist

Several lines of evidence indicate that constraining intramolecular interactions between transmembrane domains are required to maintain G protein-coupled receptors in an inactive conformation in the absence of agonist. For the glycoprotein hormone receptors, which harbor a long amino-terminal ectodomain responsible for hormone binding, it has been suggested that the ectodomain could contribute to these negative constraints. To test this hypothesis, we expressed at the surface of COS-7 cells mutants of the TSH receptor in which variable portions of the amino-terminal ectodomain are replaced by a 19-residue tag from bovine rhodopsin. Whereas none of the rhodopsin-tagged truncated mutants could be activated by saturating concentrations of TSH, the constructs with the shortest amino-terminal extension displayed increased constitutive activity toward the cAMP pathway, when compared with the wild-type holoreceptor. The shortest truncated construct was strongly activated by the introduction of mutations in transmembrane segment VI (D633A), or in the third intracellular loop (A623I) of the receptor. The magnitude of the stimulation was similar to that observed when the same mutations were introduced in the intact wild-type receptor. On the contrary, the shortest truncated construct was unaffected by activating mutations affecting residues of the extracellular loop region (I486F, I568T) or the top of transmembrane segment VII (del658-661). Together, our results are compatible with a model in which activation of the cAMP pathway by the TSH receptor involves switching of the ectodomain from a tethered inverse agonist to a true agonist.     

Cellular distribution of constitutively active mutant parathyroid hormone (PTH)/PTH-related protein receptors and regulation of cyclic adenosine 3',5'-monophosphate signaling by beta-arrestin2

PTH promotes endocytosis of human PTH receptor 1 (PTH1Rc) by activating protein kinase C and recruiting beta-arrestin2. We examined the role of beta-arrestin2 in regulating the cellular distribution and cAMP signaling of two constitutively active PTH1Rc mutants, H223R and T410P. Overexpression of a beta-arrestin2-green fluorescent protein (GFP) conjugate in COS-7 cells inhibited constitutive cAMP accumulation by H223R and T410P in a dose-dependent manner, as well as the response to PTH of both mutant and wild-type PTH1Rcs. The cellular distribution of PTH1Rc-GFP conjugates, fluorescent ligands, and ssarrestin2-GFP was analyzed by fluorescence microscopy in HEK-293T cells. In cells expressing either receptor mutant, a ligand-independent mobilization of beta-arrestin2 to the cell membrane was observed. In the absence of ligand, H223R and wild-type PTH1Rcs were mainly localized on the cell membrane, whereas intracellular trafficking of T410P was also observed. While agonists promoted beta-arrestin2-mediated endocytosis of bot PTH1Rc mutants, antagonists were rapidly internalized only with T410P. The protein kinases inhibitor, staurosporine, significantly decreased internalization of ligand-PTH1Rc mutant complexes, although the recruitment of beta-arrestin2 to the cell membrane was unaffected. Moreover, in cells expressing a truncated wild-type PTH1Rc lacking the C-terminal cytoplasmic domain, agonists stimulated translocation of beta-arrestin2 to the cell membrane followed by ligand-receptor complex internalization without associated beta-arrestin2. In conclusion, cAMP signaling by constitutively active mutant and wild-type PTH1Rcs is inhibited by a receptor interaction with beta-arrestin2 on the cell membrane, possibly leading to uncoupling from G(s)alpha. This phenomenon is independent from protein kinases activity and the receptor C-terminal cytoplasmic domain. In addition, there are differences in the cellular localization and internalization features of constitutively active PTH1Rc mutants H223R and T410P.     

Ligand-independent dimerization and activation of the oncogenic Xmrk receptor by two mutations in the extracellular domain

Overexpression of the oncogenic receptor tyrosine kinase ONC-Xmrk is the first step in the development of hereditary malignant melanoma in the fish Xiphophorus. However, overexpression of its proto-oncogene counterpart (INV-Xmrk) is not sufficient for the oncogenic function of the receptor. Compared with INV-Xmrk, the ONC-Xmrk receptor displays 14 amino acid changes, suggesting the presence of activating mutations. To identify such activating mutations, a series of chimeric and mutant receptors were studied. None of the mutations present in the intracellular domain was found to be involved in receptor activation. In the extracellular domain, we found two mutations responsible for activation of the receptor. One is the substitution of a conserved cysteine (C578S) involved in intramolecular disulfide bonding. The other is a glycine to arginine exchange (G359R) in subdomain III. Either mutation leads to constitutive dimer formation and thereby to activation of the ONC-Xmrk receptor. Besides, the presence of these mutations slows down the processing of the Xmrk receptor in the endoplasmic reticulum, which is apparent as an incomplete glycosylation.     

Constitutively active G protein-coupled receptor mutants block dictyostelium development

cAR1, a G protein-coupled receptor (GPCR) for cAMP, is required for the multicellular development of Dictyostelium. The activation of multiple pathways by cAR1 is transient because of poorly defined adaptation mechanisms. To investigate this, we used a genetic screen for impaired development to isolate four dominant-negative cAR1 mutants, designated DN1-4. The mutant receptors inhibit multiple cAR1-mediated responses known to undergo adaptation. Reduced in vitro adenylyl cyclase activation by GTPgammaS suggests that they cause constitutive adaptation of this and perhaps other pathways. In addition, the DN mutants are constitutively phosphorylated, which normally requires cAMP binding and possess cAMP affinities that are approximately 100-fold higher than that of wild-type cAR1. Two independent activating mutations, L100H and I104N, were identified. These residues occupy adjacent positions near the cytoplasmic end of the receptor's third transmembrane helix and correspond to the (E/D)RY motif of numerous mammalian GPCRs, which is believed to regulate their activation. Taken together, these findings suggest that the DN mutants are constitutively activated and block development by turning on natural adaptation mechanisms.     

Contacts between extracellular loop two and transmembrane helix six determine basal activity of the thyroid-stimulating hormone receptor

A number of alanine mutations in extracellular loop two (ECL2) of the thyroid-stimulating hormone receptor (TSHR) were found to increase or decrease basal activity when compared with the wild type receptor. K565A was identified as a mutant with decreased basal activity, and strongly impaired hormone induced signaling activity. To gain insights into how ECL2 mutants affect basal activity, we focused on constitutively activating pathogenic mutant I568V in ECL2, which exhibits elevated basal activity. Because our molecular model suggests that Ile-568 is embedded in an environment of hydrophobic residues provided by transmembrane helix bundle, we tested mutants in this region to identify potential interaction partner(s) for Ile-568. Indeed, the double mutant I568V/I640L (ECL2/TMH6) suppresses the increased basal activity exhibited by I568V alone. We suggest a spatial and functional relationship between ECL2 and TMH6 in which side chain interaction between Ile-568 and Ile-640 constrains the receptor in a conformation with low basal activity. Although the single mutant I640L exhibits basal activity lower than wild type, its differently branched and bulkier side chain complements the reduced side chain bulk in I568V, restoring wild type basal activity to the double mutant. This scenario is confirmed by the reciprocal double mutant I640V/I568L. The combination of basally increased activity of I640V and basally decreased activity of mutant I568L also restores basal activity of wild type TSHR. These and other mutant phenotypes reported here support a dynamic interface between TMH6 and ECL2. Disruption of this critical interface for signaling by introduction of mutations in TSHR can either increase or decrease basal activity.     

A single nucleotide polymorphic mutation in the human mu-opioid receptor severely impairs receptor signaling

Large scale sequencing of the human mu-opioid receptor (hMOR) gene has revealed polymorphic mutations that occur within the coding region. We have investigated whether the mutations N40D in the extracellular N-terminal region, N152D in the third transmembrane domain, and R265H and S268P in the third intracellular loop alter functional properties of the receptor expressed in mammalian cells. The N152D receptor was produced at low densities. Binding affinities of structurally diverse opioids (morphine, diprenorphine, DAMGO and CTOP) and the main endogenous opioid peptides (beta-endorphin, [Met]enkephalin, and dynorphin A) were not markedly changed in mutant receptors (<3-fold). Receptor signaling was strongly impaired in the S268P mutant, with a reduction of efficacy and potency of several agonists (DAMGO, beta-endorphin, and morphine) in two distinct functional assays. Signaling at N40D and R265H mutants was highly similar to wild type, and none of the mutations induced detectable constitutive activity. DAMGO-induced down-regulation of receptor-binding sites, following 20 h of treatment, was identical in wild-type and mutant receptors. Our data show that natural sequence variations in hMOR gene have little influence on ligand binding or receptor down-regulation but could otherwise modify receptor density and signaling. Importantly, the S268P mutation represents a loss-of-function mutation for the human mu-opioid receptor, which may have an incidence on opioid-regulated behaviors or drug addiction in vivo.     

Substitutions in the hydrophobic core of the alpha-factor receptor of Saccharomyces cerevisiae permit response to Saccharomyces kluyveri alpha-factor and to antagonist.

Mutations in the Saccharomyces cerevisiae alpha-factor receptor that lead to improved response to Saccharomyces kluyveri alpha-factor were identified and sequenced. Mutants were isolated from cells bearing randomly mutagenized receptor gene (STE2) plasmids by an in vivo screen. Five mutations lead to substitutions in hydrophobic segments in the core of the receptor (M54I, S145L, S145L-S219L, A229V, L255S-S288P). Remarkably, strains expressing these mutant receptors exhibited positive pheromone responses to desTrp1,Ala3-alpha-factor, an analog that normally blocks these responses. The M54I mutation appeared to affect only ligand specificity. The other mutations conferred additional effects on signaling or recovery. Two mutants were more sensitive to alpha-factor than wild type (S145L, A229V). One mutant was more sensitive to alpha-factor-induced cell cycle arrest initially, but then recovered more efficiently (S145L-S219L). One mutant (L255S-S288P) conferred positive pheromone responses to alpha-factor as assayed by FUS1-lacZ reporter induction, but did not display growth arrest. The hydrophobic receptor core thus appears to control activation by some ligands and to play roles in aspects of signal transduction and recovery.     

Generation of a mouse monoclonal TSH receptor antibody with stimulating activity

A Balb/c mouse was subjected to genetic immunization with a cDNA construct encoding the human thyrotropin receptor (TSHr). The immune response of the mouse resulted in the production of immunoglobulins recognizing the TSHr in three different assays: (1) flow immunocytometry (FACS) with CHO cells expressing the receptor; (2) receptor-dependent stimulation of cAMP production in the same cell line; and (3) competition with labeled TSH for binding to the receptor. One thousand hybridomas were generated from the spleen of the mouse and their supernatants were screened. A single monoclonal, IRI-SAb1, scored positive in all three assays and was studied further. It stimulated 13-fold cAMP production in TSHr-expressing CHO cells, with an EC50 in the low nanomolar range. When compared with bovine TSH, IRI-SAb1 behaved as a partial agonist. Contrary to the expectation from the characteristic of autoantibodies of Graves' patients, IRI-SAb1 recognized a linear epitope, which was localized in a segment encompassing the first 281 residues of the receptor.     

Jak2 FERM domain interaction with the erythropoietin receptor regulates Jak2 kinase activity

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement.     

Activation of the luteinizing hormone receptor following substitution of Ser-277 with selective hydrophobic residues in the ectodomain hinge region

Glycoprotein hormone receptors are G protein-coupled receptors with ligand-binding ectodomains consisting of leucine-rich repeats. The ectodomain is connected by a conserved cysteine-rich hinge region to the seven transmembrane (TM) region. Gain-of-function mutants of luteinizing hormone (LH) and thyroid-stimulating hormone receptors found in patients allowed identification of residues important for receptor activation. Based on constitutively active mutations at Ser-281 in the hinge region of the thyroid-stimulating hormone receptor, we mutated the conserved serine in the LH (S277I) and follicle-stimulating hormone receptors (S273I) and observed increased basal cAMP production and ligand affinity by mutant receptors. For the LH receptor, conversion of Ser-277 to all natural amino acids led to varying degrees of receptor activation. Hydropathy index analysis indicated that substitution of neutral serine with selective nonpolar hydrophobic residues (Leu>Val>Met>Ile) confers constitutive receptor activation whereas serine deletion or substitution with charged Arg, Lys, or Asp led to defective receptor expression. Furthermore, mutation of the angular proline near Ser-273 to flexible Gly also led to receptor activation. The findings suggest the ectodomain of glycoprotein hormone receptors constrain the TM region. Point mutations in the hinge region of these proteins, or ligand binding to these receptors, could cause conformational changes in the TM region that result in G(s) activation.     

Definition of an inhibitory juxtamembrane WW-like domain in the platelet-derived growth factor beta receptor

A variety of tumors contain activating mutations in the cytoplasmic juxtamembrane domain of the type III family of receptor-tyrosine kinases, and some constructed mutations in this domain induce ligand-independent receptor activation. To explore the role of this domain in regulation of receptor activity, we subjected the juxtamembrane domain of the murine platelet-derived growth factor (PDGF) beta receptor to alanine-scanning mutagenesis. The mutant receptors were expressed in Ba/F3 cells and tested for constitutive tyrosine phosphorylation, association with phosphatidylinositol 3'-kinase, and their ability to induce cell survival and proliferation in the absence of interleukin-3. The mutant receptors accumulated to similar levels and appeared to undergo a normal PDGF-induced increase in tyrosine phosphorylation. Alanine substitutions at numerous positions located throughout the juxtamembrane domain caused constitutive receptor activation, as did an alanine insertion in the membrane-proximal segment of the juxtamembrane domain and a six-amino acid deletion in the center of the domain. It is possible to model the PDGF receptor juxtamembrane domain as a short alpha-helix followed by a three-stranded beta-sheet very similar to the known structures of WW domains. Strikingly, the activating mutations clustered in the central portions of the first and second beta strands and along one face of the beta-sheet, whereas the loops connecting the strands were largely devoid of mutationally sensitive positions. These findings provide strong support for the model that the activating mutations in the juxtamembrane region stimulate receptor activity by disrupting an inhibitory WW-like domain.