Predicted sensitivity to endocrine therapy for stage II-III hormone receptor-positive and HER2-negative (HR+/HER2-) breast cancer before chemo-endocrine therapy.

BACKGROUND: We proposed that a test for sensitivity to the adjuvant endocrine therapy component of treatment for patients with stage II-III breast cancer (SET2,3) should measure transcription related to estrogen and progesterone receptors (SETER/PR index) adjusted for a baseline prognostic index (BPI) combining clinical tumor and nodal stage with molecular subtype by RNA4 (ESR1, PGR, ERBB2, and AURKA). PATIENTS AND METHODS: Patients with clinically high-risk, hormone receptor-positive (HR+), human epidermal growth factor receptor 2 (HER2)-negative (HR+/HER2-) breast cancer received neoadjuvant taxane-anthracycline chemotherapy, surgery with measurement of residual cancer burden (RCB), and then adjuvant endocrine therapy. SET2,3 was measured from pre-treatment tumor biopsies, evaluated first in an MD Anderson Cancer Center (MDACC) cohort (n = 307, 11 years' follow-up, U133A microarrays), cut point was determined, and then independent, blinded evaluation was carried out in the I-SPY2 trial (n = 268, high-risk MammaPrint result, 3.8 years' follow-up, Agilent-44K microarrays, NCI Clinical Trials ID: NCT01042379). Primary outcome measure was distant relapse-free survival. Multivariate Cox regression models tested prognostic independence of SET2,3 relative to RCB and other molecular prognostic signatures, and whether other prognostic signatures could substitute for SETER/PR or RNA4 components of SET2,3. RESULTS: SET2,3 added independent prognostic information to RCB in the MDACC cohort: SET2,3 [hazard ratio (HR) 0.23, P = 0.004] and RCB (HR 1.77, P < 0.001); and the I-SPY2 trial: SET2,3 (HR 0.27, P = 0.031) and RCB (HR 1.68, P = 0.008). SET2,3 provided similar prognostic information irrespective of whether RCB-II or RCB-III after chemotherapy, and in both luminal subtypes. Conversely, RCB was most strongly prognostic in cancers with low SET2,3 status (MDACC P < 0.001, I-SPY2 P < 0.001). Other molecular signatures were not independently prognostic; they could effectively substitute for RNA4 subtype within the BPI component of SET2,3, but they could not effectively substitute for SETER/PR index. CONCLUSIONS: SET2,3 added independent prognostic information to chemotherapy response (RCB) and baseline prognostic score or subtype. Approximately 40% of patients with clinically high-risk HR+/HER2- disease had high SET2,3 and could be considered for clinical trials of neoadjuvant endocrine-based treatment.

Circulating tumor DNA in neoadjuvant-treated breast cancer reflects response and survival.

BACKGROUND: Pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) is strongly associated with favorable outcome. We examined the utility of serial circulating tumor DNA (ctDNA) testing for predicting pCR and risk of metastatic recurrence. PATIENTS AND METHODS: Cell-free DNA (cfDNA) was isolated from 291 plasma samples of 84 high-risk early breast cancer patients treated in the neoadjuvant I-SPY 2 TRIAL with standard NAC alone or combined with MK-2206 (AKT inhibitor) treatment. Blood was collected at pretreatment (T0), 3 weeks after initiation of paclitaxel (T1), between paclitaxel and anthracycline regimens (T2), or prior to surgery (T3). A personalized ctDNA test was designed to detect up to 16 patient-specific mutations (from whole-exome sequencing of pretreatment tumor) in cfDNA by ultra-deep sequencing. The median follow-up time for survival analysis was 4.8 years. RESULTS: At T0, 61 of 84 (73%) patients were ctDNA positive, which decreased over time (T1: 35%; T2: 14%; and T3: 9%). Patients who remained ctDNA positive at T1 were significantly more likely to have residual disease after NAC (83% non-pCR) compared with those who cleared ctDNA (52% non-pCR; odds ratio 4.33, P = 0.012). After NAC, all patients who achieved pCR were ctDNA negative (n = 17, 100%). For those who did not achieve pCR (n = 43), ctDNA-positive patients (14%) had a significantly increased risk of metastatic recurrence [hazard ratio (HR) 10.4; 95% confidence interval (CI) 2.3-46.6]; interestingly, patients who did not achieve pCR but were ctDNA negative (86%) had excellent outcome, similar to those who achieved pCR (HR 1.4; 95% CI 0.15-13.5). CONCLUSIONS: Lack of ctDNA clearance was a significant predictor of poor response and metastatic recurrence, while clearance was associated with improved survival even in patients who did not achieve pCR. Personalized monitoring of ctDNA during NAC of high-risk early breast cancer may aid in real-time assessment of treatment response and help fine-tune pCR as a surrogate endpoint of survival.

Improved tolerability of neratinib in patients with HER2-positive early-stage breast cancer: the CONTROL trial.

BACKGROUND: Neratinib is an irreversible pan-HER tyrosine kinase inhibitor approved for extended adjuvant treatment in early-stage HER2-positive breast cancer based on the phase III ExteNET study. In that trial, in which no antidiarrheal prophylaxis was mandated, grade 3 diarrhea was observed in 40% of patients and 17% discontinued due to diarrhea. The international, open-label, sequential-cohort, phase II CONTROL study is investigating several strategies to improve tolerability. PATIENTS AND METHODS: Patients who completed trastuzumab-based adjuvant therapy received neratinib 240 mg/day for 1 year plus loperamide prophylaxis (days 1-28 or 1-56). Sequential cohorts evaluated additional budesonide or colestipol prophylaxis (days 1-28) and neratinib dose escalation (DE; ongoing). The primary end point was the incidence of grade ≥3 diarrhea. RESULTS: Final data for loperamide (L; n = 137), budesonide + loperamide (BL; n = 64), colestipol + loperamide (CL; n = 136), and colestipol + as-needed loperamide (CL-PRN; n = 104) cohorts, and interim data for DE (n = 60; completed ≥six cycles or discontinued; median duration 11 months) are available. No grade 4 diarrhea was observed. Grade 3 diarrhea rates were lower than ExteNET in all cohorts and lowest in DE (L 31%, BL 28%, CL 21%, CL-PRN 32%, DE 15%). Median number of grade 3 diarrhea episodes was one; median duration per grade 3 episode was 1.0-2.0 days across cohorts. Most grade 3 diarrhea and diarrhea-related discontinuations occurred in month 1. Diarrhea-related discontinuations were lowest in DE (L 20%, BL 8%, CL 4%, CL-PRN 8%, DE 3%). Decreases in health-related quality of life did not cross the clinically important threshold. CONCLUSIONS: Neratinib tolerability was improved with preemptive prophylaxis or DE, which reduced the rate, severity, and duration of neratinib-associated grade ≥3 diarrhea compared with ExteNET. Lower diarrhea-related treatment discontinuations in multiple cohorts indicate that proactive management can allow patients to stay on neratinib for the recommended time period. CLINICALTRIALS.GOV: NCT02400476.

WNT/ß-catenin signaling regulates mitochondrial activity to alter the oncogenic potential of melanoma in a PTEN-dependent manner.

Aberrant regulation of WNT/ß-catenin signaling has a crucial role in the onset and progression of cancers, where the effects are not always predictable depending on tumor context. In melanoma, for example, models of the disease predict differing effects of the WNT/ß-catenin pathway on metastatic progression. Understanding the processes that underpin the highly context-dependent nature of WNT/ß-catenin signaling in tumors is essential to achieve maximal therapeutic benefit from WNT inhibitory compounds. In this study, we have found that expression of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), alters the invasive potential of melanoma cells in response to WNT/ß-catenin signaling, correlating with differing metabolic profiles. This alters the bioenergetic potential and mitochondrial activity of melanoma cells, triggered through regulation of pro-survival autophagy. Thus, WNT/ß-catenin signaling is a regulator of catabolic processes in cancer cells, which varies depending on the metabolic requirements of tumors.

WNT7B mediates autocrine Wnt/ß-catenin signaling and anchorage-independent growth in pancreatic adenocarcinoma.

Developmental and cancer models show Wnt/ß-catenin-dependent signaling mediates diverse phenotypic outcomes in the pancreas that are dictated by context, duration and strength of activation. While generally assumed to be pro-tumorigenic, it is unclear to what extent dysregulation of Wnt/ß-catenin signaling impacts tumor progression in pancreatic adenocarcinoma (PDAC). In the present study, Wnt/ß-catenin activity was characterized across a spectrum of PDAC cell lines and primary tumors. Reporter and gene expression-based assays revealed wide heterogeneity in Wnt/ß-catenin transcriptional activity across PDAC cell lines and patient tumors, as well as variable responsiveness to exogenous Wnt ligand stimulation. An experimentally generated, pancreas-specific gene expression signature of Wnt/ß-catenin transcriptional activation was used to stratify pathway activation across a cohort of resected, early-stage PDAC tumors (N=41). In this cohort, higher Wnt/ß-catenin activation was found to significantly correlate with lymphvascular invasion and worse disease-specific survival (median survival time 20.3 versus 43.9 months, log-rank P=0.03). Supporting the importance of Wnt ligand in mediating autocrine Wnt signaling, Wnt/ß-catenin activity was significantly inhibited in PDAC cell lines by WLS gene silencing and the small-molecule inhibitor IWP-2, both of which functionally block Wnt ligand processing and secretion. Transcriptional profiling revealed elevated expression of WNT7B occurred in PDAC cell lines with high levels of cell autonomous Wnt/ß-catenin activity. Gene-knockdown studies in AsPC-1 and HPAF-2 cell lines confirmed WNT7B-mediated cell autonomous Wnt/ß-catenin activation, as well as an anchorage-independent growth phenotype. Our findings indicate WNT7B can serve as a primary determinant of differential Wnt/ß-catenin activation in PDAC. Disrupting the interaction between Wnt ligands and their receptors may be a particularly suitable approach for therapeutic modulation of Wnt/ß-catenin signaling in PDAC and other cancer contexts where Wnt activation is mediated by ligand expression rather than mutations in canonical pathway members.

Focal radial styloid abnormality as a manifestation of de Quervain tenosynovitis.

OBJECTIVE: de Quervain disease is a stenosing tenosynovitis of the first dorsal wrist compartment. The purpose of this study was to determine whether focal radial styloid abnormality (cortical erosion, sclerosis, or periosteal bone apposition) as shown by radiography can be an indicator of de Quervain tenosynovitis. MATERIALS AND METHODS: A retrospective review of 49 radiographs from 45 patients in whom the clinical diagnosis of de Quervain tenosynovitis was confirmed (positive findings on Finkelstein's test) and 64 radiographs from 62 asymptomatic patients was carried out independently by two musculoskeletal radiologists in a blinded fashion. Findings on radiographs were assessed for focal radial styloid abnormality and assigned a diagnostic grade (1, definitely normal; 2, probably normal; 3, equivocal; 4, probably abnormal; 5, definitely abnormal). Receiver operating characteristic curves were constructed and compared. Kappa statistics for interobserver and intraobserver variability were calculated. RESULTS: The presence of focal radial styloid abnormality correlated significantly with the presence of de Quervain tenosynovitis (p < 0.05). The areas under the receiver operating characteristic curves for each reviewer equaled 0.71 and 0.76. Kappa values for interobserver variability equaled 0.44 (moderate agreement), and intraobserver variability equaled 0.62 (substantial agreement). CONCLUSION: Focal radial styloid abnormality is an indicator of de Quervain stenosing tenosynovitis of the wrist.

Identification of the sites phosphorylated by cyclic AMP-dependent protein kinase on the beta 2 subunit of L-type voltage-dependent calcium channels.

Voltage-dependent L-type calcium (Ca) channels are heteromultimeric proteins that are regulated through phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrated that the beta 2 subunit was a substrate for PKA in intact cardiac myocytes through back-phosphorylation experiments. In addition, a heterologously expressed rat beta 2a subunit was phosphorylated at two sites in vitro by purified PKA. This beta 2a subunit contains two potential consensus sites for PKA-mediated phosphorylation at Thr164 and Ser591. However, upon mutation of both of these residues to alanines, the beta 2a subunit remained a good substrate for PKA. The actual sites of phosphorylation on the beta 2a subunit were identified by phosphopeptide mapping and microsequencing. Phosphopeptide maps of a bacterially expressed beta 2a subunit demonstrated that this subunit was phosphorylated similarly to the beta 2 subunit isolated from heart tissue and that the phosphorylation sites were contained in the unique C-terminal region. Microsequencing identified three serine residues, each of which conformed to loose consensus sites for PKA-mediated phosphorylation. Mutation of these residues to alanines resulted in the loss of the PKA-mediated phosphorylation of the beta 2a subunit. The results suggest that phosphorylation of the beta 2a subunit by PKA occurs at three loose consensus sites for PKA in the C-terminus and not at either of the two strong consensus sites for PKA. The results also highlight the danger of assuming that consensus sites represent actual sites of phosphorylation. The actual sites of PKA-mediated phosphorylation are conserved in most beta 2 subunit isoforms and thus represent potential sites for regulation of channel activity. The sites phosphorylated by PKA are not substrates for protein kinase C (PKC), as the mutated beta 2 subunits lacking PKA sites remained good substrates for PKC.

Complexes of the alpha1C and beta subunits generate the necessary signal for membrane targeting of class C L-type calcium channels.

In the present study, we investigated the role of channel subunits in the membrane targeting of voltage-dependent L-type calcium channel complexes. We co-expressed the calcium channel pore-forming alpha1C subunit with different accessory beta subunits in HEK-tsA201 cells and examined the subcellular localization of the channel subunits by immunohistochemistry using confocal microscopy and whole-cell radioligand binding studies. While the pore-forming alpha1C subunit exhibited perinuclear staining when expressed alone, and several of the wild-type and mutant beta subunits also exhibited intracellular staining, co-expression of the alpha1C subunit with either the wild-type beta2a subunit, a palmitoylation-deficient beta2a(C3S/C4S) mutant or three other nonpalmitoylated beta isoforms (beta1b, beta3, and beta4 subunits) resulted in the redistribution of both the alpha1C and beta subunits into clusters along the cell surface. Furthermore, the redistribution of calcium channel complexes to the plasma membrane was observed when alpha1C was co-expressed with an N- and C-terminal truncated mutant beta2a containing only the central conserved regions. However, when the alpha1C subunit was co-expressed with an alpha1 beta interaction-deficient mutant, beta2aBID-, we did not observe formation of the channels at the plasma membrane. In addition, an Src homology 3 motif mutant of beta2a that was unable to interact with the alpha1C subunit also failed to target channel complexes to the plasma membrane. Interestingly, co-expression of the pore-forming alpha1C subunit with the largely peripheral accessory alpha2 delta subunit was ineffective in recruiting alpha1C to the plasma membrane, while co-distribution of all three subunits was observed when beta2a was co-expressed with the alpha1C and alpha2 delta subunits. Taken together, our results suggested that the signal necessary for correct plasma membrane targeting of the class C L-type calcium channel complexes is generated as a result of a functional interaction between the alpha1 and beta subunits.

Post-translational modifications of beta subunits of voltage-dependent calcium channels.

Different post-translational modifications of Ca channel beta subunits have been identified. Recent studies have characterized the palmitoylation of the Ca channel beta2a subunit, as well as one effect of this modification on channel function. The potential importance of palmitoylation on other channel properties is discussed. Other studies have addressed the role of phosphorylation of beta subunits in the regulation of voltage-dependent Ca channels. Phosphorylation of beta subunits by second messenger-activated protein kinases, as well as by unidentified protein kinases, may affect interactions between channel subunits and other aspects of channel function. The differential modification of Ca channel beta subunit isoforms by post-translational events likely results in diversely regulated channels with unique properties.

Membrane targeting of L-type calcium channels. Role of palmitoylation in the subcellular localization of the beta2a subunit.

In this study, we report that palmitoylation was a critical determinant of the subcellular localization of the rat beta2a subunit of voltage-dependent calcium channels. Immunohistochemical staining of transfected cells revealed that a palmitoylation-deficient beta2a subunit exhibited a diffuse intracellular staining pattern, in contrast to the plasma membrane distribution seen with the wild-type beta2a subunit. Unexpectedly, mutations in regions distal to the palmitoylation sites at Cys3 and Cys4 affected palmitoylation of the beta2a protein. Mutations in an src homology 3 motif of the beta2a subunit affected both palmitoylation and subcellular localization of the beta2a protein. A mutation in the beta interaction domain, which disrupted interactions between the expressed alpha1 and beta subunits, also resulted in a decreased palmitoylation and diffuse intracellular localization of the beta2a protein. Studies of chimeric proteins revealed that the 16-amino acid N terminus of the beta2a subunit was sufficient to confer palmitoylation to the nonpalmitoylated beta1b and beta3 isoforms. However, palmitoylation of chimeric beta subunits was by itself insufficient to restore the plasma membrane localization observed with the wild-type beta2a protein. Treatment of transfected cells with brefeldin A increased the amount of palmitic acid incorporated in the beta2a protein, suggesting that palmitoylation of beta2a occurs during or shortly after protein synthesis. Two other beta2 variants, the rabbit beta2a and beta2b, which lack the palmitoylation sties at Cys3 and Cys4, exhibited a diffuse intracellular staining pattern and were not palmitoylated.

Sorcin associates with the pore-forming subunit of voltage-dependent L-type Ca2+ channels.

Intracellular Ca2+ release in muscle is governed by functional communication between the voltage-dependent L-type Ca2+ channel and the intracellular Ca2+ release channel by processes that are incompletely understood. We previously showed that sorcin binds to cardiac Ca2+ release channel/ryanodine receptors and decreases channel open probability in planar lipid bilayers. In addition, we showed that sorcin antibody immunoprecipitates ryanodine receptors from metabolically labeled cardiac myocytes along with a second protein having a molecular weight similar to that of the alpha1 subunit of cardiac L-type Ca2+ channels. We now demonstrate that sorcin biochemically associates with cardiac and skeletal muscle L-type Ca2+ channels specifically within the cytoplasmically oriented C-terminal region of the alpha1 subunits, providing evidence that the second protein recovered by sorcin antibody from cardiac myocytes was the 240-kDa L-type Ca2+ channel alpha1 subunit. Anti-sorcin antibody immunoprecipitated full-length alpha1 subunits from cardiac myocytes, C2C12 myotubes, and transfected non-muscle cells expressing alpha1 subunits. In contrast, the anti-sorcin antibody did not immunoprecipitate C-terminal truncated forms of alpha1 subunits that were detected in myotubes. Recombinant sorcin bound to cardiac and skeletal HIS6-tagged alpha1 C termini immobilized on Ni2+ resin. Additionally, anti-sorcin antibody immunoprecipitated C-terminal fragments of the cardiac alpha1 subunit exogenously expressed in mammalian cells. The results identified a putative sorcin binding domain within the C terminus of the alpha1 subunit. These observations, along with the demonstration that sorcin accumulated substantially during physiological maturation of the excitation-contraction coupling apparatus in developing postnatal rat heart and differentiating C2C12 muscle cells, suggest that sorcin may mediate interchannel communication during excitation-contraction coupling in heart and skeletal muscle.

Identification and subcellular localization of the subunits of L-type calcium channels and adenylyl cyclase in cardiac myocytes.

The properties of cardiac L-type channels have been well characterized electrophysiologically, and many such studies have demonstrated that the channels are regulated by a cAMP-dependent pathway. However, the subunit composition of native cardiac L-type calcium channels has not been completely defined. Furthermore, a very important question exists regarding the status of the C-terminal domain of the pore-forming alpha1 subunit, as this domain has the potential to be the target of protein kinases but may be truncated as a result of post-translational processing. In the present studies, the alpha1C and beta2 subunits were identified by subunit-specific antibodies after partial purification from heart membranes, or immunoprecipitation from cardiac myocytes. Both the beta2 and the full-length alpha1C subunits were found to be expressed and co-localized in intact cardiac myocytes along T-tubule membranes. Using a quantitative antibody binding analysis, we demonstrated that the majority of the alpha1C subunits in intact cardiac myocytes appear to be full-length. In addition, we observed that adenylyl cyclase is localized in a pattern similar to the channel subunits in cardiac myocytes. Taken together, our results provide new insights into the structural basis for understanding the regulation of L-type calcium channels by a cAMP-mediated signaling pathway.

Localization of lipoprotein(a) in a monkey model of rapid neointimal growth.

Lipoprotein(a) [Lp(a)] has been proposed as a restenosis risk factor, but it is not known if Lp(a) is present in the injured arterial wall during the initial neointimal growth. The purpose of this study was to determine if Lp(a) is incorporated into the vessel wall during rapid neointimal formation after arterial injury in primates. In this model, distention of the iliac artery with an angioplasty catheter caused focal breaks in the internal elastic lamina (IEL) in 80% of the vessels and extensive IEL fragmentation with medial disruption in 20% of the vessels. Neointimal growth was noted in all injured arteries; thrombus formation was noted in 40% of the vessels. Based on morphometric measurements, injured arteries had neointimal areas of 0.41 +/- 0.05 (n = 4) and 0.83 +/- 0.23 (n = 6) mm2 at 14 and 28 days after injury, respectively. Control arteries had an intact IEL and a monolayer of intimal cells. Lp(a) localization was examined histologically by using a mouse monoclonal anti-Lp(a) antibody. Lp(a), found in all injured arteries, was localized primarily in the neointima in 50% of the vessels. In the subset of vessels with evidence of thrombus formation, intense Lp(a) immunostaining was associated with the thrombus. Lp(a) was specific to injured arteries as uninjured vessels did not stain. In addition, staining was not seen with a negative control, a nonspecific mouse IgG1 antibody. The presence of Lp(a) at the site of rapid neointimal growth supports a role for this lipoprotein in the response to vascular injury after balloon angioplasty.

Identification of palmitoylation sites within the L-type calcium channel beta2a subunit and effects on channel function.

The hydrophilic beta2a subunit of the L-type calcium channel was recently shown to be a membrane-localized, post-translationally modified protein (Chien, A. J., Zhao, X. L., Shirokov, R. E., Puri, T. S., Chang, C. F., Sun, D. D., Rios, E., and Hosey, M. M. (1995) J. Biol. Chem. 270, 30036-30044). In this study, we demonstrate that the rat beta2a subunit was palmitoylated through a hydroxylamine-sensitive thioester linkage. Palmitoylation required a pair of cysteines in the N terminus, Cys3 and Cys4; mutation of these residues to serines resulted in mutant beta2a subunits that were unable to incorporate palmitic acid. Interestingly, a palmitoylation-deficient beta2a mutant still localized to membrane particulate fractions and was still able to target functional channel complexes to the plasma membrane similar to wild-type beta2a. However, channels formed with a palmitoylation-deficient beta2a subunit exhibited a dramatic decrease in ionic current per channel, indicating that although mutations eliminating palmitoylation did not affect channel targeting by the beta2a subunit, they were important determinants of channel modulation by the beta2a subunit. Three other known beta subunits that were analyzed were not palmitoylated, suggesting that palmitoylation could provide a basis for the regulation of L-type channels through modification of a specific beta isoform.

The Tridimensional Personality Questionnaire in depression: state versus trait issues.

The goal of our study was to examine the stability over time of the personality measures defined by Cloninger's Tridimensional Personality Questionnaire (TPQ) in patients with major depression. We conducted a retrospective chart review of 63 outpatients who had participated in one of three double-blind antidepressant trials. All subjects had completed the TPQ upon entry and upon termination of the 12-week trials. Statistically significant decrements in harm avoidance scores were observed in those subjects whose depression improved, regardless of gender or duration of presenting depressive episode. No significant changes were observed in the other two dimensions (novelty seeking and reward dependence). Nonresponders as a group had no statistically significant decreases in any TPQ dimension or subscales. While novelty seeking and reward dependence remain relatively independent of mood, harm avoidance appears to be a measure which is elevated during depressive states and which changes with improvement of depression. These findings are consistent with those of other investigators.

Structure and regulation of L-type calcium channels a current assessment of the properties and roles of channel subunits.

L-type Ca channels are complex heteromultimeric proteins that play important roles in the cardiovascular system. Recent studies have revealed new insights into how the pore-forming α(1) subunits interact with accessory subunits to produce functional Ca channels. The function of L-type Ca channels is often regulated by receptor-mediated signal transduction events that are thought to result in the phosphorylation of proteins that comprise the Ca channels. Although the molecular events underlying phosphorylation based regulation have been intensely investigated with the use of electrophysiological approaches, surprisingly few details are known about the biochemical events involved, and many questions remain unanswered. © 1996, Elsevier Science Inc. (Trends Cardiovasc Med 1996;6:265-273).

Roles of a membrane-localized beta subunit in the formation and targeting of functional L-type Ca2+ channels.

We report several unexpected findings that provide novel insights into the properties and interactions of the alpha 1 and beta subunits of dihydropyridine-sensitive L-type channels. First, the beta 2a subunit was expressed as multiple species of 68-72 kDa; the 70-72-kDa species arose from post-translational modification. Second, cell fractionation and immunocytochemical studies indicated that the hydrophilic beta 2a subunit, when expressed alone, was membrane-localized. Third, the beta 2a subunit increased the membrane localization of the alpha 1 subunit and the number of cells expressing L-type Ca2+ currents, without affecting the total amount of the expressed alpha 1C subunit. Expression of maximal currents in alpha 1C/beta 2a cotransfected cells paralleled the time course of expression of the beta subunit. Taken together, these results suggest that the beta subunit plays multiple roles in the formation, stabilization, targeting, and modulation of L-type channels.

Effects of transposable elements on the expression of the forked gene of Drosophila melanogaster.

The products of the forked gene are involved in the formation and/or maintenance of a temporary fibrillar structure within the developing bristle rudiment of Drosophila melanogaster. Mutations in the forked locus alter this structure and result in aberrant development of macrochaetae, microchaetae and trichomes. The locus has been characterized at the molecular level by walking, mutant characterization and transcript analysis. Expression of the six forked transcripts is temporally restricted to mid-late pupal development. At this time, RNAs of 6.4, 5.6, 5.4, 2.5, 1.9 and 1.1 kilobases (kb) are detected by Northern analysis. The coding region of these RNAs has been found to be within a 21-kb stretch of genomic DNA. The amino terminus of the proteins encoded by the 5.4- and 5.6-kb forked transcripts contain tandem copies of ankyrin-like repeats that may play an important role in the function of forked-encoded products. The profile of forked RNA expression is altered in seven spontaneous mutations characterized during this study. Three forked mutations induced by the insertion of the gypsy retrotransposon contain a copy of this element inserted into an intron of the gene. In these mutants, the 5.6-, 5.4- and 2.5-kb forked mRNAs are truncated via recognition of the polyadenylation site in the 5' long terminal repeat of the gypsy retrotransposon. These results help explain the role of the forked gene in fly development and further our understanding of the role of transposable elements in mutagenesis.