Epidermal growth factor receptor association with β1-adrenergic receptor is mediated via its juxtamembrane domain

β1-adrenergic receptor (β1AR)-mediated transactivation of epidermal growth factor receptor (EGFR) engages downstream signaling events that impact numerous cellular processes including growth and survival. While association of these receptors has been shown to occur basally and be important for relaying transactivation-specific intracellular events, the mechanism by which they do so is unclear and elucidation of which would aid in understanding the consequence of disrupting their interaction. Using fluorescence resonance energy transfer (FRET) and immunoprecipitation (IP) analyses, we evaluated the impact of C-terminal truncations of EGFR on its ability to associate with β1AR. While loss of the last 230 amino acid C-terminal phosphotyrosine-rich domain did not disrupt the ability of EGFR to associate with β1AR, truncation of the entire intracellular domain of EGFR resulted in almost complete loss of its interaction with β1AR, suggesting that either the kinase domain or juxtamembrane domain (JMD) may be required for this association. Treatment with the EGFR antagonist gefitinib did not prevent β1AR-EGFR association, however, treatment with a palmitoylated peptide encoding the first 20 amino acids of the JMD domain (JMD-A) disrupted β1AR-EGFR association over time and prevented β1AR-mediated ERK1/2 phosphorylation, both in general and specifically in association with EGFR. Conversely, neither a mutated JMD-A peptide nor a palmitoylated peptide fragment consisting of the subsequent 18 amino acids of the JMD domain (JMD-B) were capable of doing so. Altogether, the proximal region of the JMD of EGFR is responsible for its association with β1AR, and its disruption prevents β1AR-mediated transactivation, thus providing a new tool to study the functional consequences of disrupting β1AR-EGFR downstream signaling.     

C-terminal domains of Na(+)/H(+) exchanger isoform 3 are involved in the basal and serum-stimulated membrane trafficking of the exchanger

When expressed either in polarized epithelial cells or in fibroblasts, two Na(+)/H(+) exchanger isoforms, NHE1 and NHE3, have different subcellular distributions. Using a quantitative cell surface biotinylation technique, we found PS120 cells target approximately 90% of mature NHE1 but only 14% of NHE3 to the cell surface, and this pattern occurs irrespective of NHE protein expression levels. In this study, we examined surface fractions of NHE3 C-terminal truncation mutants to identify domains involved in the targeting of NHE3. Removing the C-terminal 76 amino acids doubled surface fractions to 30% of total and doubled the V(max) from 1300 to 2432 microM H(+)/s. Removal of another 66 amino acids increased surface levels to 55% of total with an increase in the V(max) to 5794 microM H(+)/s. Surface fractions did not change with a further 105 amino acid truncation. We postulated that inhibition of the basal recycling of NHE3 could result in the surface accumulation of the NHE3 truncations. Accordingly, we found that, unlike wild-type NHE3, the truncations were shown to internalize poorly and were not affected by PI3 kinase inhibition. However, while the truncations demonstrated reduced basal recycling, they retained the same serum response as full-length NHE3, with a mobilization of approximately 10% of total NHE to the surface. We conclude that basal recycling of NHE3 is controlled by endocytic determinants contained within its C-terminal 142 amino acids and that serum-mediated exocytosis is independently regulated through a different part of the protein.     

Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants.

Overexpression and autocrine activation of the epidermal growth factor receptor (EGF-R) cause transformation of cultured cells and correlate with tumor progression in cancer patients. Dimerization and transphosphorylation are crucial events in the process by which receptors with tyrosine kinase activity generate normal and transforming cellular signals. Interruption of this process by inactive receptor mutants offers the potential to inhibit ligand-induced cellular responses. Using recombinant retroviruses, we have examined the effects of signalling-incompetent EGF-R mutants on the growth-promoting and transforming potential of ligand-activated, overexpressed wild-type EGF-R and the v-erbB oncogene product. Expression of a soluble extracellular EGF-R domain had little if any effect on the growth and transformation of NIH 3T3 cells by either tyrosine kinase. However, both a kinase-negative EGF-R point mutant (HERK721A) and an EGF-R lacking 533 C-terminal amino acids efficiently inhibited wild-type EGF-R-mediated, de novo DNA synthesis and cell transformation in a dose-dependent manner. Furthermore, coexpression with the v-erbBES4 oncogene product in NIH 3T3 cells resulted in transphosphorylation of the HERK721A mutant receptor and reduced soft-agar colony growth but had no effect in a focus formation assay. These results demonstrate that signalling-defective receptor tyrosine kinase mutants differentially interfere with oncogenic signals generated by either overexpressed EGF-R or the retroviral v-erbBES4 oncogene product.     

Independent functions for the N- and C-termini in the overlap region of tropomyosin

Tropomyosin (TM) is a coiled-coil that binds head-to-tail along the helical actin filament. The ends of 284-residue tropomyosins are believed to overlap by about nine amino acids. The present study investigates the function of the N- and C-terminal overlap regions. Recombinant tropomyosins were produced in Escherichia coli in which nine amino acids were truncated from the N-terminal, C-terminal, or both ends of striated muscle alpha-tropomyosin (TM9a) and TM2 (TM9d), a nonmuscle alpha-tropomyosin expressed in many cells. The two isoforms are identical except for the C-terminal 27 amino acids encoded by exon 9a (striated) or exon 9d (TM2). Removal of either end greatly reduces the actin affinity of both tropomyosins in all conditions and the cooperativity with which myosin promotes tropomyosin binding to actin in the open state. N-Terminal truncations generally are more deleterious than C-terminal truncations. With TM9d, truncation of the N-terminus is as deleterious as both for myosin S1-induced binding. None of the TM9d variants binds well to actin with troponin (+/-Ca(2+)). TM9a with the truncated N-terminus binds more weakly to actin with troponin (-Ca(2+)) than when the C-terminus is removed but more strongly than when both ends are removed; the actin binding of all three forms is cooperative. The results show that the ends of TM9a, though important, are not required for cooperative function and suggest they have independent functions beyond formation of an overlap complex. The nonadditivity of the TM9d truncations suggests that the ends may primarily function as a complex in this isoform. A surprising result is that all variants bound with the same affinity, and noncooperatively, to actin saturated with myosin S1. Evidently, end-to-end interactions are not required for high-affinity binding to acto-myosin S1.     

Ligand-independent dimerization of oncogenic v-erbB products involves covalent interactions.

Mutant v-erbB products of avian c-erbB1 have previously been used to correlate structural domains of the receptor encoded by this proto-oncogene with tissue-specific transformation potential. In these studies, deletion of the ligand-binding domain of the receptor has been shown to be required for transformation of erythroblasts, fibroblasts, and endothelial cells. It has, therefore, been postulated that deletion of this domain results in an allosteric change in the receptor analogous to the ligand-bound state of the epidermal growth factor receptor; i.e., it induces a receptor conformation that is constitutively active with respect to mitogenic signaling. While oncogenic v-erbB products have been shown to be expressed on the cell surface of both fibroblasts and erythroblasts, no comprehensive analysis of the oligomeric potential of these products has been conducted. Since the first event known to follow epidermal growth factor binding to its receptor is oligomerization, and receptor dimerization has been correlated with mitogenic signaling, we have carefully analyzed the ability of several v-erbB products to oligomerize in the three target cell types transformed by these oncogenes. In this report, we demonstrate the v-erbB products can efficiently homodimerize in all three target tissues, that this dimerization is ligand independent and occurs at the cell surface, and that there is no apparent correlation between v-erbB dimerization and transformation of avian fibroblasts. Furthermore, both oncogenic and nononcogenic v-erbB products can heterodimerize with the native c-erbB1 product in chicken embryo fibroblasts, suggesting that heterodimerization between v-erB and native c-erbB1 is not sufficient to result in c-erbB1-mediated sarcomagenesis.     

Antibodies against a synthetic peptide as a probe for the kinase activity of the avian EGF receptor and v-erbB protein

The transforming protein v-erbB of avian erythroblastosis virus (AEV) displays extensive sequence homology with the presumptive protein-tyrosine kinase domain of the human EGF receptor and with the src protein-tyrosine kinase family of oncogenes. However, no kinase activity has previously been demonstrated for the v-erbB protein. Here antibodies generated against a synthetic peptide from the C terminus of human EGF receptor are shown to immunoprecipitate the EGF receptor from human and avian cells, as well as the v-erbB proteins from AEV-transformed cells that become phosphorylated on tyrosine residues upon the addition of gamma-32P-ATP. The immunoprecipitates are also able to phosphorylate exogenous tyrosine-containing substrates. Hence, it is likely that both avian EGF receptor and v-erbB proteins are protein tyrosine-specific protein kinases. Since the kinase activity of v-erbB protein cannot be regulated by EGF, it is proposed that the tyrosine protein kinase function of v-erbB may be constitutively activated.     

Herstatin, an autoinhibitor of the human epidermal growth factor receptor 2 tyrosine kinase, modulates epidermal growth factor signaling pathways resulting in growth arrest

Herstatin is an autoinhibitor of the ErbB family consisting of subdomains I and II of the human epidermal growth factor receptor 2 (ErbB-2) extracellular domain and a novel C-terminal domain encoded by an intron. Herstatin binds to human epidermal growth factor receptor 2 and to the epidermal growth factor receptor (EGFR), blocking receptor oligomerization and tyrosine phosphorylation. In this study, we characterized several early steps in EGFR activation and investigated downstream signaling events induced by epidermal growth factor (EGF) and by transforming growth factor alpha (TGF-alpha) in NIH3T3 cell lines expressing EGFR with and without herstatin. Herstatin expression decreased EGF-induced EGFR tyrosine phosphorylation and delayed receptor down-regulation despite receptor occupancy by ligand with normal binding affinity. Akt stimulation by EGF and TGF-alpha, but not by fibroblast growth factor 2, was almost completely blocked in the presence of herstatin. Surprisingly, EGF and TGF-alpha induced full activation of MAPK in duration and intensity and stimulated association of the EGFR with Shc and Grb2. Although MAPK was fully stimulated, herstatin expression prevented TGF-alpha-induced DNA synthesis and EGF-induced proliferation. The herstatin-mediated uncoupling of MAPK from Akt activation was also observed in Chinese hamster ovary cells co-transfected with EGFR and herstatin. These findings show that herstatin expression alters EGF and TGF-alpha signaling profiles, culminating in inhibition of proliferation.     

The effect of activating mutations on dimerization, tyrosine phosphorylation and internalization of the macrophage colony stimulating factor receptor.

Oncogenic activation of the macrophage colony stimulating factor (M-CSF) receptor (c-Fms) requires mutation or truncation of the carboxyl terminus and specific amino acid substitutions in or near the fourth immunoglobulin (Ig)-like loop in the extracellular domain. Using a murine c-Fms system, we investigated the effect of C-terminal truncation, substitutions at amino acids 301 and 374 in the fourth Ig-like loop of the extracellular domain, or the combined mutations on individual steps in receptor activation. The mutations at amino acids 301 and 374 were necessary, but not sufficient, for receptor dimerization in the absence of M-CSF. Only receptors with a truncated C-terminus as well as the extracellular domain mutations dimerized efficiently in the absence of M-CSF, suggesting that the C-terminus of c-Fms also regulates receptor oligomerization. Truncation of the C-terminus alone did not cause receptor dimerization and did not activate the kinase enzymatic activity. Thus, truncation of the C-terminus did not activate receptor monomers in cis. Receptors with both a truncated C-terminus and the extracellular domain mutations underwent ligand-independent aggregation, transphosphorylation, and phosphorylation of cellular proteins, followed by rapid internalization and degradation. These results suggest that M-CSF binding to c-Fms initiates activation by inducing conformational changes in both the cytoplasmic C-terminal domain and the fourth Ig-like loop of the extracellular domain, leading to the formation of stable receptor dimers.     

Temperature-sensitive mutants of avian erythroblastosis virus: surface expression of the erbB product correlates with transformation

The v-erbB gene of avian erythroblastosis virus (AEV) codes for an integral plasma membrane glycoprotein, gp74erbB. Expression of gp74erbB and its intracellular precursors, gp66erbB and gp68erbB, has been studied in cells transformed by two temperature-sensitive mutants of AEV. After shift to 42 degrees C, the processing of gp68erbB is blocked in tsAEV-transformed, but not in wtAEV-transformed, erythroblasts and fibroblasts. In addition, gp74erbB disappears from the surface of tsAEV cells within 12 hr after shift. Thus tsAEV mutants probably bear a lesion in v-erbB that affects the maturation and subcellular localization of gp74erbB. The tsAEV erythroblasts, when "committed" to differentiation by a pulse-shift to 42 degrees C, reexpress gp74erbB during terminal differentiation at 36 degrees C. This suggests that tsAEV erythroblasts become insensitive to the transforming functions of gp74erbB at a certain stage of differentiation.     

The amino-terminal 14 amino acids of v-src can functionally replace the extracellular and transmembrane domains of v-erbB.

The retroviral oncogene v-erbB encodes a truncated form of the receptor for epidermal growth factor, an integral membrane protein-tyrosine kinase. By contrast, the oncogene v-src encodes a protein-tyrosine kinase that is a peripheral membrane protein. The morphologies and spectra of cells transformed by these two oncogenes differ. In an effort to identify the functional determinant(s) of these differences, we constructed and tested first deletion mutants of v-erbB and then chimeras between v-src and v-erbB. As reported previously, the absence of any membrane anchorage eliminated transformation by v-erbB. Anchorage of the cytoplasmic kinase domain of v-erbB to membranes with amino-terminal portions of the v-src protein permitted transformation. The phenotype and spectrum of transformation were those expected for v-erbB rather than for v-src. The transforming chimeras lost their biological activity if the signal for myristylation at the amino terminus of v-src was compromised by mutation. Biochemical fractionations revealed a correlation between transforming activity and the association of chimeric gene products with the membrane fraction of the cell. For reasons not yet apparent, the combined presence of membrane anchorage domains of v-src, and the transmembrane domain of v-erbB in the same chimera typically (but not inevitably) impeded transformation. Our results suggest that the specificity of transformation by v-erbB resides in the selection of substrates by the cytoplasmic domain of the gene product. The protein retains access to those substrates even when anchored to the membrane in the manner of a peripheral rather than a transmembrane protein.     

Functional heterogeneity of proto-oncogene tyrosine kinases: the C terminus of the human epidermal growth factor receptor facilitates cell proliferation.

Previous reports have indicated that the C termini of the membrane-associated tyrosine kinases encoded by c-src and c-fms proto-oncogenes have a negative effect on their biological activity and that this effect is mediated by their C-terminal tyrosine residue. To determine whether this was true for the human epidermal growth factor (EGF) receptor, which is also a membrane-associated tyrosine kinase proto-oncogene, we have constructed two premature termination mutants, dc19 and dc63, that delete the C-terminal 19 and 63 amino acids, respectively, from the human full-length receptor (hEGFR). The smaller deletion removes the C-terminal tyrosine residue, while the larger deletion removes the two most C-terminal tyrosines; similar deletions are found in v-erbB. As previously shown for the gene encoding the full-length EGF receptor, the two C-terminal mutants induced EGF-dependent focal transformation and anchorage-independent growth of NIH 3T3 cells. However, both dc19 and dc63 were quantitatively less efficient than the gene encoding the full-length receptor, with dc63 being less active than dc19. Although the C-terminal mutants displayed lower biological activity than the gene encoding the full-length receptor, the mutant receptors were found to be similar in several respects to the full-length receptor. These parameters included receptor localization, stability in the absence of EGF, receptor half-life in the presence of EGF, EGF binding, extent of EGF-dependent autophosphorylation in vitro, and EGF-dependent phosphorylation of an exogenous substrate in vitro. Therefore, the C-terminal 63 amino acids of the human receptor have no detectable influence on EGF-dependent early events. We conclude that in contrast     

Inactivation of parkin by oxidative stress and C-terminal truncations: a protective role of molecular chaperones

Loss of parkin function is linked to autosomal recessive juvenile parkinsonism. Here we show that proteotoxic stress and short C-terminal truncations induce misfolding of parkin. As a consequence, wild-type parkin was depleted from a high molecular weight complex and inactivated by aggregation. Similarly, the pathogenic parkin mutant W453Stop, characterized by a C-terminal deletion of 13 amino acids, spontaneously adopted a misfolded conformation. Mutational analysis indicated that C-terminal truncations exceeding 3 amino acids abolished formation of detergent-soluble parkin. In the cytosol scattered aggregates of misfolded parkin contained the molecular chaperone Hsp70. Moreover, increased expression of chaperones prevented aggregation of wild-type parkin and promoted folding of the W453Stop mutant. Analyzing parkin folding in vitro indicated that parkin is aggregation-prone and that its folding is dependent on chaperones. Our study demonstrates that C-terminal truncations impede parkin folding and reveal a new mechanism for inactivation of parkin.     

Functional consequences of N- or C-terminal deletions of the delta subunit of chloroplast ATP synthase

Mutagenesis was used to generate seven truncation mutants of the spinach (Spinacia oleracea) chloroplast ATP synthase delta subunit lacking 5, 11, 17, or 35 amino acid residues from the N-terminus or 3, 9, or 15 residues from the C-terminus. Interactions between these mutants and all other subunits of the chloroplast ATPase were investigated by a yeast two-hybrid system. The results indicate that the N-terminal deletions mainly affected interactions between the delta subunit and the other part of CF(1), but did not significantly affect interactions with the CF(0) sector. In contrast, C-terminal truncations of the delta subunit mainly affected its interaction with the CF(0) sector and caused little impairment in interactions with the other part of CF(1). The conformation of the delta subunit C-terminal domain seems to be more sensitive to the truncations, as shown by minimal expression driven by C-terminal deleted (nine residues) mutants. Further studies showed C-terminal truncations of the delta subunit greatly impaired its ability to restore cyclic photophosphorylation in NaBr vesicles, whereas N-terminal truncations had little effect on the ability of delta to plug the CF(0) channel. None of the mutants impaired ATP hydrolysis by CF(1).     

Mapping the multimerization domains of the Gag protein of yeast retrotransposon Ty1.

The two-hybrid system was used to define regions of the Ty1 Gag protein responsible for multimerization. Gag truncations lacking the first 146 or the last 97 amino acids (Gag is 440 amino acids in length) interact. A severely C-terminally truncated molecule (lacking the last 207 amino acids) was the smallest truncation to interact, suggesting that some protein-protein interactions between Gag molecules are mediated through the first 233 amino acids. However, an internal deletion of amino acids 147 to 233 does not abolish Gag-Gag interaction, indicating that more than one region can mediate Gag interaction. Surprisingly, we found that a truncation lacking the last 97 amino acids interacts with itself but not with full-length Gag. This is apparently due to an artifact of the two-hybrid assay, since these same molecules coassemble with wild-type Gag into Ty1 virus-like particles.     

Identification of sequences required for inhibition of oncogene-mediated transformation by pp32.

Oncogenic potential in prostate cancer is modulated in part by alternative use of genes of the pp32 family. This family includes the tumor suppressor pp32, expressed in normal tissue, and the pro-oncogenic genes pp32r1 and pp32r2 that are found principally in neoplastic cells. At the protein level, pp32, pp32r1, and pp32r2 are approximately 90% identical, yet they subsume opposite functions. In this study, we identify the region of pp32 associated with the ability to inhibit oncogene-mediated transformation in a rat embryo fibroblast system, an in vitro correlate of tumor-suppressive activity. Deletion and truncation analysis define a region spanning pp32 amino acids 150-174 as absolutely required for inhibition of transformed foci elicited by RAS and MYC. Comparison of pp32 with the pp32r1 sequence by moving averages of sequence identity reveals divergence over this region; pp32r2 also differs in this region through truncation after pp32 amino acid 131. The deletion experiments and the experiments of nature therefore converge to demonstrate that tumor-suppressive functions of pp32 reside in amino acids 150-174. Identification of this minimal tumor-suppressive region should help elaborate the pathways and mechanisms through which pp32 family members exert their functions.     

Mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation, and infectivity

The membrane-spanning domain (MSD) of a number of retroviral transmembrane (TM) glycoproteins, including those from the human and simian immunodeficiency viruses (HIV and SIV), have been predicted to contain a charged arginine residue. The wild-type SIV TM glycoprotein is 354 amino acids long. The entire putative cytoplasmic domain of SIV (amino acids 193 to 354) is dispensable for virus replication in vitro, and such truncation-containing viruses are capable of reaching wild-type titers after a short delay. We show here that further truncation of eight additional amino acids to TM185 results in a protein that lacks fusogenicity but is, nevertheless, efficiently incorporated into budding virions. By analyzing a series of nonsense mutations between amino acids 193 and 185 in Env expression vectors and in the SIVmac239 proviral clone, a region of the SIV TM that contains the minimum requirement for glycoprotein-mediated cell-to-cell fusion and that for virus replication was identified. Virus entry and infectivity were evident in truncations to a minimum of 189 amino acids, whereas cell-cell fusion was observed for a protein of only 187 amino acids. Glycoprotein was efficiently incorporated into budding virions in truncations up to 185 amino acids, indicating that such proteins are membrane anchored and are transported to the cell surface. However, truncation of the TM to 180 amino acids resulted in a protein that displays a transport defect and may be retained in the endoplasmic reticulum. Based on our analyses of these mutants, an alternative model for the MSD of SIV is proposed. Our model suggests that membrane-imbedded charged residues can be neutralized by side-chain interactions with lipid polar head groups. As a consequence, the membrane-spanning region can be reduced by more than a helical turn. This new model accounts for the ability of truncations within the predicted MSD to remain membrane anchored and maintain biological activity.     

Structure of PICK1 and other PDZ domains obtained with the help of self-binding C-terminal extensions

PDZ domains are protein-protein interaction modules that generally bind to the C termini of their target proteins. The C-terminal four amino acids of a prospective binding partner of a PDZ domain are typically the determinants of binding specificity. In an effort to determine the structures of a number of PDZ domains we have included appropriate four residue extensions on the C termini of PDZ domain truncation mutants, designed for self-binding. Multiple truncations of each PDZ domain were generated. The four residue extensions, which represent known specificity sequences of the target PDZ domains and cover both class I and II motifs, form intermolecular contacts in the expected manner for the interactions of PDZ domains with protein C termini for both classes. We present the structures of eight unique PDZ domains crystallized using this approach and focus on four which provide information on selectivity (PICK1 and the third PDZ domain of DLG2), binding site flexibility (the third PDZ domain of MPDZ), and peptide-domain interactions (MPDZ 12th PDZ domain). Analysis of our results shows a clear improvement in the chances of obtaining PDZ domain crystals by using this approach compared to similar truncations of the PDZ domains without the C-terminal four residue extensions.     

Identification of the XPG region that causes the onset of Cockayne syndrome by using Xpg mutant mice generated by the cDNA-mediated knock-in method

In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause early onset of Cockayne syndrome (CS) in some patients (XPG/CS). The CS-causing mutations in such patients all produce truncated XPG proteins. To test the hypothesis that the CS phenotype, with characteristics such as growth retardation and a short life span in XPG/CS patients, results from C-terminal truncations, we constructed mutants with C-terminal truncations in mouse XPG (Xpg) (from residue D811 to the stop codon [XpgD811stop] and deletion of exon 15 [Xpg Delta ex15]). In the XpgD811stop and Xpg Delta ex15 mutations, the last 360 and 183 amino acids of the protein were deleted, respectively. To generate Xpg mutant mice, we devised the shortcut knock-in method by replacing genomic DNA with a mutated cDNA fragment (cDNA-mediated knock in). The control mice, in which one-half of Xpg genomic DNA fragment was replaced with a normal Xpg cDNA fragment, had a normal growth rate, a normal life span, normal sensitivity to UV light, and normal DNA repair ability, indicating that the Xpg gene partially replaced with the normal cDNA fragment retained normal functions. The XpgD811stop homozygous mice exhibited growth retardation and a short life span, but the Xpg Delta ex15 homozygous mice did not, indicating that deletion of the last 360 amino acids results in the CS phenotype but deletion of the last 183 amino acids does not. The XpgD811stop homozygous mice, however, exhibited a slightly milder CS phenotype than did the Xpg null mutant mice, indicating that the XpgD811stop protein still retains some Xpg function that affects the severity of the CS phenotype.     

Regulation of Gi by the CB1 cannabinoid receptor C-terminal juxtamembrane region: structural requirements determined by peptide analysis

A CB1 cannabinoid receptor peptide fragment from the C-terminal juxtamembrane region autonomously inhibits adenylyl cyclase activity in a neuroblastoma membrane preparation. The cannabinoid receptor antagonist, SR141716A, failed to block the response. The peptide was able to evoke the response in membranes from Chinese hamster ovary (CHO) cells that do not express the CB1 receptor. These studies are consistent with a direct activation of Gi by the peptide. To test the importance of a BXBXXB sequence, Lys403 was acetylated, resulting in a peptide having similar affinity but reduced efficacy. N-Terminal truncation of Arg401 resulted in a 6-fold loss of affinity, which was not further reduced by sequential truncation of up to the first seven amino acids, four of which are charged. N-Terminal-truncated peptides exhibited maximal activity, suggesting that Gi activation can be conferred by the remaining amino acids. Truncation of the C-terminal Glu417 or substitution of Glu417 by a Leu or of Arg401 by a Norleucine reduced activity at 100 microM. The C-terminal juxtamembrane peptide was constrained to a loop peptide by placement of Cys residues at both terminals and disulfide coupling. This modification reduced the affinity 3-fold but yielded near-maximal efficacy. Blocking the Cys termini resulted in a loss of efficacy. Circular dichroism spectropolarimetry revealed that all C-terminal juxtamembrane peptide analogues exist in a random coil conformation in an aqueous environment. A hydrophobic environment (trifluoroethanol) failed to induce alpha-helix formation in the C-terminal juxtamembrane peptide but did so in less active peptides. The anionic detergent sodium dodecyl sulfate induced alpha-helix formation in all analogues except the loop peptide, where it induces a left-handed PII conformation. It is concluded that alpha-helix formation is not required for Gi activation.     

C-terminal amino acid residues are required for the folding and cholesterol binding property of perfringolysin O, a pore-forming cytolysin.

Perfringolysin O (theta-toxin) is a pore-forming cytolysin whose activity is triggered by binding to cholesterol in the plasma membrane. The cholesterol binding activity is predominantly localized in the beta-sheet-rich C-terminal half. In order to determine the roles of the C-terminal amino acids in theta-toxin conformation and activity, mutants were constructed by truncation of the C terminus. While the mutant with a two-amino acid C-terminal truncation retains full activity and has similar structural features to native theta-toxin, truncation of three amino acids causes a 40% decrease in hemolytic activity due to the reduction in cholesterol binding activity with a slight change in its higher order structure. Furthermore, both mutants were found to be poor at in vitro refolding after denaturation in 6 M guanidine hydrochloride, resulting in a dramatic reduction in cholesterol binding and hemolytic activities. These activity losses were accompanied by a slight decrease in beta-sheet content. A mutant toxin with a five-amino acid truncation expressed in Escherichia coli is recovered as a further truncated form lacking the C-terminal 21 amino residues. The product retains neither cholesterol binding nor hemolytic activities and shows a highly disordered structure as detected by alterations in the circular dichroism and tryptophan fluorescence spectra. These results show that the C-terminal region of theta-toxin has two distinct roles; the last 21 amino acids are involved to maintain an ordered overall structure, and in addition, the last two amino acids at the C-terminal end are needed for protein folding in vitro, in order to produce the necessary conformation for optimal cholesterol binding and hemolytic activities.