Single Transmembrane Receptor Research Articles

Notch signaling and cardiac repair

Notch signaling is critical for proper heart development and recently has been reported to participate in adult cardiac repair. Notch resides at the cell surface as a single pass transmembrane receptor, transits through the cytoplasm following activation, and acts as a transcription factor upon entering the nucleus. This dynamic and widespread cellular distribution allows for potential interactions with many signaling and binding partners. Notch displays temporal as well as spatial versatility, acting as a strong developmental signal, controlling cell fate determination and lineage commitment, and playing a pivotal role in embryonic and adult stem cell proliferation and differentiation. This review serves as an update of recent literature addressing Notch signaling in the heart, with attention to findings from noncardiac research that provide clues for further interpretation of how the Notch pathway influences cardiac biology. Specific areas of focus include Notch signaling in adult myocardium following pathologic injury, the role of Notch in cardiac progenitor cells with respect to differentiation and cardiac repair, crosstalk between Notch and other cardiac signaling pathways, and emerging aspects of noncanonical Notch signaling in heart.

Upregulation of fibroblast growth factor receptor 2 and 3 in the late stages of fetal lung development in the nitrofen rat model

Nitrofen model of congenital diaphragmatic hernia (CDH) has been widely used to investigate the pathogenesis of pulmonary hypoplasia (PH). Fibroblast growth factor (FGF) signaling pathway plays a fundamental role in fetal lung development. FGF7 and FGF10, which are critical for lung morphogenesis, have been reported to be downregulated in nitrofen-induced PH. FGF signaling is mediated by a family of four single transmembrane receptors, FGFR1-4. FGFR2 and FGFR3 have been shown to be expressed predominantly in the late stages of developing lungs. In addition, the upregulation of FGFR2 gene expression has been associated with severe defects in lung development and resulted in arrested alveologenesis similar to PH seen in the nitrofen model. Furthermore, FGFR3(-/-)FGFR4(-/-) double mutants showed thinner mesenchyme and larger air spaces. We designed this study to test the hypothesis that FGFR gene expression is upregulated in the late stages of lung development in the nitrofen CDH model. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Cesarean section was performed and fetuses were harvested on D18 and D21. Fetal lungs were divided into three groups: control, nitrofen without CDH [CDH(-)], and nitrofen with CDH [CDH(+)] (n = 24 at each time-point). Pulmonary gene expression levels of FGFR1-4 were analyzed by real-time RT-PCR. Immunohistochemistry was also performed to evaluate protein expression/distribution at each time-point. The relative messenger RNA expression levels of pulmonary FGFR2 and FGFR3 on D21 were significantly increased in CDH(-) (6.38 ± 1.93 and 7.84 ± 2.86, respectively) and CDH(+) (7.09 ± 2.50 and 7.25 ± 3.43, respectively) compared to controls (P < 0.05 and P < 0.01, respectively), whereas no significant alteration was observed on D18. There were no differences in FGFR1 and FGFR4 expression at both time-points. Increased immunoreactivity of FGFR2 and FGFR3, mainly in the distal epithelium and mesenchyme, was observed in the nitrofen-induced hypoplastic lungs on D21 compared to controls. Upregulation of FGFR2 and FGFR3 pulmonary gene expression in the late stages of fetal lung development may disrupt FGFR-mediated alveologenesis resulting in PH in the CDH model.

G Protein-coupled Receptor Kinases Phosphorylate LRP6 in the Wnt Pathway

Wnt ligands conduct their functions in canonical Wnt signaling by binding to two receptors, the single transmembrane low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and seven transmembrane (7TM) Frizzled receptors. Subsequently, phosphorylation of serine/threonine residues within five repeating signature PPPSP motifs on LRP6 is responsible for LRP6 activation. GSK3β, a cytosolic kinase for phosphorylation of a downstream effector β-catenin, was proposed to participate in such LRP6 phosphorylation. Here, we report a new class of membrane-associated kinases for LRP6 phosphorylation. We found that G protein-coupled receptor kinases 5 and 6 (GRK5/6), traditionally known to phosphorylate and desensitize 7TM G protein-coupled receptors, directly phosphorylate the PPPSP motifs on single transmembrane LRP6 and regulate Wnt/LRP6 signaling. GRK5/6-induced LRP6 activation is inhibited by the LRP6 antagonist Dickkopf. Depletion of GRK5 markedly reduces Wnt3A-stimulated LRP6 phosphorylation in cells. In zebrafish, functional knock-down of GRK5 results in reduced Wnt signaling, analogous to LRP6 knock-down, as assessed by decreased abundance of β-catenin and lowered expression of the Wnt target genes cdx4, vent, and axin2. Expression of GRK5 rescues the diminished β-catenin and axin2 response caused by GRK5 depletion. Thus, our findings identify GRK5/6 as novel kinases for the single transmembrane receptor LRP6 during Wnt signaling.

Targeting the RET Pathway in Thyroid Cancer

The RET (rearranged during transfection) protooncogene encodes a single pass transmembrane receptor that is expressed in cells derived from the neural crest and the urogenital tract. As part of a cell-surface complex, RET binds glial derived neurotrophic factor (GDNF) ligands in conjunction with GDNF-family alpha co-receptors (GFRalpha). Ligand-induced activation induces dimerization and tyrosine phosphorylation of the RET receptor with downstream activation of several signal transduction pathways. Activating germline RET mutations play a central role in the development of the multiple endocrine neoplasia (MEN) syndromes MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC) and also in the development of the congenital abnormality Hirschsprung's disease. Approximately 50% of patients with sporadic MTC have somatic RET mutations, and a significant portion of papillary thyroid carcinomas result from chromosomal inversions or translocations, which activate RET (RET/PTC oncogenes). The RET protooncogene has a significant place in cancer prevention and treatment. Timely thyroidectomy in kindred members who have inherited a mutated RET allele, characteristic of MEN2A, MEN2B, or FMTC, can prevent MTC, the most common cause of death in these syndromes. Also, recently developed molecular therapeutics that target the RET pathway have shown activity in clinical trials of patients with advanced MTC, a disease for which there has been no effective therapy.

Change of the Notch1 Expression Level in Lymphoid Malignancies.

Abstract 4425Notch gene family encodes single pass transmembrane receptors able to transducer intercellular signals involved in cell-fate determination. Notch signaling is the leading causes to the development of malignant tumors. Notch pathway plays a role in haematopoiesis affecting both stem cells and committed progenitors. The expression of Notch1 in thymic stromal cells and in developing thymocytes is consistent with Notch function. Notch1 is turn on before T-cell commitment along the lymphoid development; a variety of model systems indicates that Notch1 signaling is required for the earliest stages of T-cell commitment in the thymus and that, in its absence, cells develop toward the B-lineage. Overexpression of Notch1 gene was found in T-ALL, however, little is known the expression pattern of Notch1 gene in the different lymphoid malignancies. In the present study, the expression level of Notch1 gene was analyzed in patients with lymphoid malignancies. Real-time PCR with SYBR Green I technique was used for detecting Notch1 gene expression level in peripheral blood mononuclear cells of 43 patients with lymphoid malignancies [including 7 cases with T-ALL, 6 cases with T-NHL, 9 cases with B-ALL, 5 cases with B-CLL, 16 cases with B-NHL] and 20 healthy individuals. β2-microglobulin gene (β2M) was used as an endogenous reference. Relative changes in Notch1 gene expression level were used by the 2-ΔCt×100% ×100% method. The expression level of Notch1 in both T-cell lymphoid malignancies group (Median: 0.897%) and B-cell lymphoid malignancies group (Median: 0.726%) was significant higher than those in the healthy control (Median: 0.288%) (P<0.01, P<0.01). Moreover, the expression level of Notch1 was different in the five types of lymphoid malignancies. That is, the highest in the B-CLL group, secondary in the T-NHL group, the lowest in the B-NHL group. And, it is not significant different between the B-NHL and healthy control groups. In conclusions, Overexpression of Notch1 gene was found not only in T-ALL, but also in T-NHL and B-cell leukemia and suggests that the abnormal overexpression pattern of Notch1 might play an important role in the pathogenesis of lymphoid malignancies. Down-regulation of Notch1 might be considered as target therapeutic strategy for lymphocytic malignancies. Disclosures:Zheng:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Yang:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Chen:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Zhong:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding. Li:The study was supported by grants from National Natural Science Foundation of China (No. 30871091): Research Funding.

The receptor RAGE: Bridging inflammation and cancer

The receptor for advanced glycation end products (RAGE) is a single transmembrane receptor of the immunoglobulin superfamily that is mainly expressed on immune cells, neurons, activated endothelial and vascular smooth muscle cells, bone forming cells, and a variety of cancer cells. RAGE is a multifunctional receptor that binds a broad repertoire of ligands and mediates responses to cell damage and stress conditions. It activates programs responsible for acute and chronic inflammation, and is implicated in a number of pathological diseases, including diabetic complications, stroke, atheriosclerosis, arthritis, and neurodegenerative disorders. The availability of Rage knockout mice has not only advanced our knowledge on signalling pathways within these pathophysiological conditions, but also on the functional importance of the receptor in processes of cancer. Here, we will summarize molecular mechanisms through which RAGE signalling contributes to the establishment of a pro-tumourigenic microenvironment. Moreover, we will review recent findings that provide genetic evidence for an important role of RAGE in bridging inflammation and cancer.

The Membrane Proximal Extracellular Domain of Human hGC-B Folds Independently

Human Guanylyl Cyclase B (hGC-B) is a single-transmembrane receptor protein which upon binding C-type natriuretic peptide (CNP) to its extracellular domain catalyzes the intracellular conversion of GTP to the second messenger cGMP. cGMP in turn affects various physiological processes such as smooth muscle contraction, cell proliferation, phototransduction, and salt as well as fluid homeostasis. The 3-dimensional binding site of the peptide hormone is unknown, and the binding mechanism is not yet understood. Therefore, a model of the C-terminal moiety of the extracellular domain of human GC-B containing the potential binding site was derived from the crystal structure of (GC-A). The selected protein sequence was provided with an N-terminal TEV-cleavage site and fused with a 109 aa thioredoxin-tag and a hexahistidine-tag. The identity of the purified 25 kDa protein was confirmed by protein mass fingerprint and its secondary structure was determined by CD- and NMR-spectroscopy. The protein proved to be properly folded with the observed secondary structure matching the predicted secondary structure and the homologous structure in the extracellular domain of GC-A. Size exclusion chromatography confirmed the monomeric state of P-hGC-B.

Characterization of the Tetratricopeptide-Containing Domain of BUB1, BUBR1, and PP5 Proves That Domain Amphiphilicity over Amino Acid Sequence Specificity Governs Protein Adsorption and Interfacial Activity

The tetratricopeptide motif repeat (TPR) is an alpha-helix-turn-alpha-helix motif that typically mediates protein-protein and, in some cases, protein-lipid interactions. Because of its success, this motif has been preserved through evolution and can be identified in proteins of a wide range of functions in lower and higher organisms. The N-terminal region of BUB1, BUBR1, and protein phosphatase 5 (PP5) contains tandem arrangements of the TPR motif. BUB1 and BUBR1 are conserved multidomain protein kinases that play a key role in the mitotic checkpoint, the mechanism that ensures the synchrony of chromosome segregation. PP5 is an enzyme that targets a wide range of protein substrates including single transmembrane receptors and mammalian cryptochromes. The N-terminal TPR domain of PP5 regulates the activity of the C-terminal catalytic domain through direct interaction with protein and lipid molecules. We portray the biophysical and biochemical properties of the tandem arrangements of the TPR motif of BUB1, BUBR1, and PP5 using far-UV spectroscopy, solution X-ray scattering, null ellipsometry, surface rheology measurements, and Brewster angle microscopy (BAM) observations. We show that, despite the low amino acid sequence conservation and different function, the TPR motif repeats of the three proteins exhibit similar interfacial properties including adsorption kinetics, high surface activity, and the formation of stable, rigid films at the air/water interface. Our studies demonstrate that domain amphiphilicity is of higher importance than amino acid sequence specificity in the determination of protein adsorption and interfacial activity.

Cancer stem cells

The Sixteenth International Symposium of the Hiroshima Cancer Seminar (HCS) Foundation was held on October 22 2006 at the International Conference Center, Hiroshima. The symposium consisted of 10 special lectures and 23 free paper presentations for a poster session. About 230 people were present and actively discussed cancer stem cells. Prior to this symposium, an Open Lecture to the public by HCS and the Japan Society for Dying with Dignity was held on October 21 where Shigehito Yamawaki (Hiroshima University, Hiroshima) and Kazuko Hamanaka (Hamanaka Dermatological Clinic, Hiroshisma) spoke about Psycho-oncology and Breast Cancer to more than 260 people. Eiichi Tahara (Hiroshima Cancer Seminar Foundation), Chairman of the Organizing Committee of the Sixteenth International Symposium, and Chairman of the HCS Foundation, gave an opening address. Tahara introduced a brief background and the purpose of this series of symposia. Since the establishment of the HCS Foundation in 1992, annual international symposia are organized to create an opportunity for basic scientists and clinical researchers to exchange ideas for cancer research, cancer prevention and cancer therapy. This year, the organizing committee planned to explore the important issue of cancer stem cells. Stem cells have a critical role not only in the generation of new populations of normal cells but also in the development of tumors. The balance between self-renewal and differentiation is strictly regulated to maintain normal stem cell pools and to generate the required supply of fully differentiated cells. Recent evidence has suggested that a subset of cancer cells within the tumor, so-called cancer stem cells, may drive the growth and progression of the tumor. Eradication of cancer stem cells may be essential to a cure for cancer. Advances in our knowledge that regulate proliferation, self-renewal, survival and differentiation of cancer stem cells and normal stem cells may shed light on the mechanism that leads to cancer and perhaps improve cancer treatment. The participants will be able to profit by exchanging ideas and learning from the informative presentations and discussions, and will contribute to our understanding of stem cells in relation to cancer development and treatment.

Role of the Ram Domain and Ankyrin Repeats on Notch Signaling and Activity in Cells of Osteoblastic Lineage

Notch proteins belong to a family of single pass transmembrane receptors that are activated after interactions with the membrane-bound ligands Delta and Jagged/Serrate. We determined the pathways responsible for the inhibitory effects of Notch on osteoblastogenesis and the contributions of the RAM domain and ankyrin repeats to this process in cells of the osteoblastic lineage. Notch receptors play a role in osteoblast differentiation. Activation of Notch results in its cleavage and the release of its intracellular domain (NICD), which interacts with the CBF1/RBP-Jkappa, Suppressor of Hairless, Lag-1 (CSL) family of transcription factors. The interaction is presumably mediated by the RBP-Jkappa-associated module (RAM) of NICD, although the role of the ankyrin repeats is uncertain. To determine the contributions of the RAM domain and ankyrin repeats to the inhibitory effects of Notch on osteoblastogenesis, ST-2 and MC3T3-E1 cells were transfected or transduced with vectors expressing NICD, RAM (NICD DeltaRAM), and ankyrin (NICD DeltaANK) deletion mutants. Notch increased the transactivation of transiently transfected 12xCSL-Luc constructs, containing 12 repeats of an RBP-Jkappa/CSL binding site, and of the hairy and E (spl) (HES)-1 promoter. Deletion of the ankyrin repeats resulted in the loss of 12xCSL-Luc and HES-1 promoter transactivation, whereas deletion of the RAM domain caused a partial loss of 12xCSL-Luc and sustained HES-1 promoter transactivation. NICD overexpression inhibited osteocalcin mRNA levels and alkaline phosphatase activity in ST-2 cells, and deletion of the ankyrin repeats, and to a lesser extent of the RAM domain, resulted in loss of the NICD inhibitory effect. NICD inhibited Wnt signaling and deletion of ankyrin repeats or the RAM domain restored Wnt signaling activity. The RAM domain and ankyrin repeats are required for Notch signaling and activity, and the CSL pathway is central to the inhibitory effect of Notch on osteoblastogenesis.

Pharmacological Characterization of Novel α-Calcitonin Gene-Related Peptide (CGRP) Receptor Peptide Antagonists That Are Selective for Human CGRP Receptors

Human alpha-calcitonin gene-related peptide (CGRP) is a 37-residue neuropeptide that produces a variety of cardiovascular and other effects via activation of specific CGRP receptors that produce cAMP. Functional CGRP receptors are a heterodimeric complex composed of the heptahelical calcitonin receptor-like receptor and the single transmembrane receptor activity-modifying protein 1. Based on the known structures of the antagonist CGRP((8-37)) and the human CGRP receptor, we designed novel CGRP receptor peptide antagonists with modifications to promote high affinity and selectivity for human CGRP receptors. Antagonist affinity (K(B)) at CGRP receptors was determined using the mouse thoracic aorta and human SK-N-MC cells. In aorta, CGRP((8-37)), [N-alpha-benzoyl]human alpha-CGRP((8-37)) [bzl-CGRP((8-37))], and [N-alpha-benzoyl-His(10)-benzyl]human alpha-CGRP((8-37)) [bzl-bn-CGRP((8-37))] caused rightward shifts in the concentration-response relaxation curve for CGRP with K(B) values of 1000, 88, and 50 nM, respectively. In human SK-N-MC cells, CGRP((8-37)), bzl-CGRP((8-37)), and bzl-bn-CGRP((8-37)) caused rightward shifts in the concentration-response curve for CGRP-stimulated cAMP production with K(B) values of 797, 15, and 0.63 nM, respectively. Thus, CGRP((8-37)) had the same affinity for human and mouse CGRP receptors, whereas bzl-CGRP((8-37)) and bzl-bn-CGRP((8-37)) displayed 6- and 80-fold higher affinities, respectively, for human CGRP receptors. In addition, the selectivity of the antagonists for human CGRP receptors was highly correlated with the antagonist hydrophobicity index. These relatively high-affinity, species-selective peptide antagonists provide novel tools to differentiate structural and functional features that are unique to the human CGRP receptor. Thus, these analogs may be useful compounds for development of drugs to treat migraine headache and other cardiovascular diseases.

Identification of two functional guanylin receptors in eel: Multiple hormone-receptor system for osmoregulation in fish intestine and kidney

Guanylyl cyclase C (GC-C) is a single transmembrane receptor for a family of intestinal hormones, guanylins. In the eel, we previously identified three guanylins, whose gene expression was enhanced in the intestine after transfer from fresh water to seawater. However, only limited information is available about the structure and function of their receptor(s). In the present study, we cloned full-length cDNAs encoding two isoforms of GC-C, named GC-C1 and GC-C2, from eel intestine. The predicted GC-C proteins consisted of extracellular ligand-binding domain, membrane-spanning domain, kinase-like domain and cyclase catalytic domain, in which GC-C-specific sequences were largely conserved. Phylogenetic analyses showed that the cloned membrane GCs are grouped with the GC-C of other vertebrates but not with GC-A and GC-B. However, eel GC-Cs appear to have undergone unique structural evolution compared with other GC-Cs. The three eel guanylins (guanylin, uroguanylin and renoguanylin), but not eel atrial natriuretic peptide, stimulated cGMP production dose-dependently in COS cells expressing either of the cloned cDNAs, providing functional support for assignment as eel guanylin receptors. The potency order for cGMP production was uroguanylin > guanylin ⩾ renoguanylin for GC-C1; guanylin ⩾ renoguanylin > uroguanylin for GC-C2. The distinctive ligand selectivity was consistent with the low homology (53%) of the extracellular domain of the two GC-Cs compared with that observed for other domains (74–90%). Both GC-C genes were expressed in the alimentary tract (esophagus, stomach and intestine) and kidney, and their expression was higher in the intestine of seawater-adapted eels than that of freshwater eels just as observed with the guanylin genes. However, the expression of the receptor genes was unchanged for 24 h after transfer of eels from fresh water to seawater or vice versa, showing slower response of the receptors to salinity changes than their ligands. Collectively, the multiple guanylin—GC-C system may be involved as a paracrine factor in seawater adaptation at the intestine and kidney of the eel.

SorLA Signaling by Regulated Intramembrane Proteolysis

The single-transmembrane receptor SorLA/LR11 contains binding domains typical for lipoprotein receptors and a VPS10 domain, which binds the neuropeptide head-activator. This undecapeptide enhances proliferation of neuronal precursor cells in a SorLA-dependent manner. Using specific inhibitors we found previously that head activator activates shedding of SorLA by the metalloprotease TACE close to the transmembrane domain releasing the large extra-cellular part of the receptor. Here we show that the remaining COOH-terminal membrane fragment of SorLA is processed by gamma-secretase. Inhibition of gamma-secretase by specific inhibitors or overexpression of dominant negative presenilin mutants and knock out of the presenilin genes led to accumulation of the SorLA membrane fragment and also of full-length SorLA in the membrane. In an in vitro assay we observed the gamma-secretase-dependent release of the two soluble cleavage products, the SorLA cytoplasmic domain and the SorLA beta-peptide. These processing steps are reminiscent of a novel signaling pathway that has been described for the notch receptor. Here, the notch cytoplasmic domain is released into the cytoplasm by the gamma-secretase and migrates to the nucleus where it acts as a transcriptional regulator. In parallel we found that a fusion protein of the released cytoplasmic tail of SorLA with EGFP located to the nucleus only if the nuclear localization signal of SorLA was intact. In a reporter gene assay the cytoplasmic domain of SorLA acted as a transcriptional activator indicating that SorLA might directly regulate transcription after activation by gamma-secretase.

Notch receptor encodes two structurally separable functions in Drosophila: A genetic analysis

The Notch gene of Drosophila encodes a single transmembrane receptor that plays a central role in the process of lateral inhibition. This process results in the selection of individual mesodermal and neural precursors during the development of the muscular and nervous systems. The activation of Notch during lateral inhibition is mediated by the transmembrane ligand Delta (Dl) and effected by the transcription factor Suppressor of Hairless (Su(H)). The same functional cassette plays a role in other processes, in particular, the development and patterning of the wing. Genetic analysis has suggested that, in addition to the Su(H)-dependent pathway, Notch can signal in an Su(H)-independent manner. This process seems to be tightly associated with signalling by Wingless, a member of the Wnt family of signalling molecules. Here, we have analyzed further the possibility that the Notch protein encodes two different functions. To do so, we have studied the activities and genetic properties of different Notch receptors bearing deletions of specific regions of the intracellular and the extracellular domains in different developmental processes, and have sought to correlate the activity of these mutant proteins with those of existing mutants in Notch. Our results support the existence of at least two different activities of Notch each of which can be associated with specific structural domains.

β-Arrestin Regulates Notch Abundance

β-Arrestin, well known for its role in G protein-coupled receptor regulation, is also being recognized for its roles in regulating other types of receptors. Mukherjee et al. report that Drosophila β-arrestin, Kurtz (Krz), is involved in controlling the abundance of the receptor Notch. Notch is a single transmembrane receptor that is cleaved in response to ligand binding, releasing a fragment that translocates to the nucleus to regulate transcription. Krz was found in two different screens for proteins that interacted with the Notch regulator and with putative E3 ubiquitin ligase Deltex (Dx). In flies, loss of Krz function led to increased Notch abundance. Overexpression of both Krz and Dx produced Notch loss-of-function phenotypes and reduced Notch protein abundance. In transfected Drosophila S2 cells, Krz and Dx together promoted ubiquitination of Notch. Notch signaling is highly sensitive to gene dosage effects, and β-arrestin appears to be one component that contributes to this sensitivity. — NG

β-Arrestin Regulates Notch Abundance

β-Arrestin, well known for its role in G protein-coupled receptor regulation, is also being recognized for its roles in regulating other types of receptors. Now Mukherjee et al. report that Drosophila β-arrestin, Kurtz (Krz), is involved in controlling the abundance of the receptor Notch, which is a single transmembrane receptor that is cleaved in response to ligand binding, releasing a fragment that translocates to the nucleus to regulate transcription. Krz was found in two different screens for proteins that interacted with the Notch regulator and putative E3 ubiquitin ligase Deltex (Dx). Coimmunoprecipitation experiments and colocalization experiments confirmed the interaction of Krz and Dx and further showed that Notch and these two proteins form a ternary complex. When Krz and Dx were transfected into Drosophila S2 cells, the two proteins colocalized in intracellular vesicles and, in fly wings, overexpression of the two proteins led to depletion of Notch from Dx-positive vesicles. In flies, loss of Krz function led to increased Notch abundance and, in a context-dependent manner, this produced increased Notch signaling. Overexpression of both Krz and Dx produced Notch loss-of-function phenotypes and reduced Notch protein abundance. In transfected Drosophila S2 cells, Krz and Dx together promoted ubiquitination of Notch, which was detectable in the presence of proteasome inhibitors. Notch signaling is highly sensitive to gene dosage effects, and β-arrestin appears to be one component that contributes to this sensitivity. Questions that remain include whether Krz recruits additional E3 ubiquitin ligases to the internalized receptor or whether Krz alters the E3 ligase activity of Dx to promote Notch ubiquitination (see Shenoy and Lefkowitz for commentary). A. Mukherjee, A. Veraksa, A. Bauer, C. Rosse, J. Camonis, S. Artavanis-Tsakonas, Regulation of Notch signalling by non-visual β-arrestin. Nat. Cell Biol. 7 , 1191-1201 (2005). [PubMed] S. K. Shenoy, R. J. Lefkowitz, Receptor regulation: β-arrestin moves up a notch. Nat. Cell Biol. 7 , 1159-1161 (2005). [PubMed]

Disulfide Linkages and a Three-Dimensional Structure Model of the Extracellular Ligand-binding Domain of Guanylyl Cyclase C

Guanylyl cyclase C (GC-C) is a single-transmembrane receptor that is specifically activated by endogenous ligands, including guanylin, and the exogenous ligand, heat-stable enterotoxin. Using combined HPLC separation and MS analysis techniques the positions of the disulfide linkages in the extracellular ligand-binding domain (ECD) of GC-C were determined to be between Cys7-Cys94, Cys72-Cys77, Cys101-Cys128 and Cys179-Cys226. Furthermore, a three-dimensional structural model of the ECD was constructed by homology modeling, using the structure of the ECD of GC-A as a template (van den Akker et al., 2000, Nature, 406: 101-104) and the information of the disulfide linkages. Although the GC-C model was similar to the known structure of GC-A, importantly its ligand-binding site appears to be located on the quite different region from that in GC-A.

Notch1 is essential for postnatal hair follicle development and homeostasis

Notch genes encode evolutionarily conserved large, single transmembrane receptors, which regulate many cell fate decisions and differentiation processes during fetal and postnatal life. Multiple Notch receptors and ligands are expressed in both developing and adult epidermis and hair follicles. Proliferation and differentiation of these two ectodermal-derived structures have been proposed to be controlled in part by the Notch pathway. Whether Notch signaling is involved in postnatal hair homeostasis is currently unknown. Here, we investigate and compare the role of the Notch1 receptor during embryonic hair follicle development and postnatal hair homeostasis using Cre-loxP based tissue specific and inducible loss-of-function approaches. During embryonic development, tissue-specific ablation of Notch1 does not perturb formation and patterning of hair follicle placodes. However, Notch1 deficient hair follicles invaginate prematurely into the dermis. Embryonic as well as postnatal inactivation of Notch1 shortly after birth or in adult mice results in almost complete hair loss followed by cyst formation. The first hair cycle of Notch1 deficient mice is characterized by shortened anagen and a premature entry into catagen. These data show that Notch1 is essential for late stages of hair follicle development during embryogenesis as well as for post-natal hair follicle development and hair homeostasis.

Internalization and trafficking of guanylyl (guanylate) cyclase/natriuretic peptide receptor A is regulated by an acidic tyrosine-based cytoplasmic motif GDAY

We have identified a GDAY motif in the C-terminal domain of guanylyl cyclase (guanylate cyclase)/NPRA (natriuretic peptide receptor A) sequence, which serves a dual role as an internalization signal and a recycling signal. To delineate the role of the GDAY motif in receptor internalization and sequestration, we mutated Gly920, Asp921 and Tyr923 to alanine residues (GDAY/AAAA) in the NPRA cDNA sequence. The cDNAs encoding wild-type and mutant receptors were transfected in HEK-293 cells (human embryonic kidney 293 cells). The internalization studies of ligand-receptor complexes revealed that endocytosis of 125I-ANP by HEK-293 cells expressing G920A, Y923A or GDAY/AAAA mutant receptor was decreased by almost 50% (P<0.001) when compared with cells expressing the wild-type receptor. However, the effect of D921A mutation on receptor internalization was minimal. Ligand-mediated down-regulation of G920A, Y923A and GDAY/AAAA mutant receptors was decreased by 35-40% when compared with wild-type NPRA. Subsequently, the recycling of internalized D921A and GDAY/AAAA mutant receptors from the intracellular pool was decreased by more than 40+/-4% when compared with wild-type NPRA. Recycling of G920A and Y923A mutant receptors was also decreased, but to a significantly lesser extent compared with the D921A or GDAY/AAAA mutant receptors. We conclude that the Gly920 and Tyr923 residues within the GDAY consensus motif are necessary for internalization, and that residue Asp921 is important for recycling of NPRA. The current results provide new evidence for a dual role of the GDAY sequence motif in ligand-mediated internalization, recycling and down-regulation of a single-transmembrane receptor protein NPRA.

The Transmembrane Domains of ErbB Receptors do not Dimerize Strongly in Micelles

The epidermal growth factor receptors (erbB) constitute an important class of single pass transmembrane receptors involved in the transduction of signals important for cell proliferation and differentiation. Receptor association is a key event in the signal transduction process, but the molecular basis of this interaction is not fully understood. Previous biochemical and genetic studies have suggested that the single transmembrane helices of these receptor proteins might play a role in stabilizing the receptor complexes. To determine if the erbB transmembrane domains could provide a driving force to stabilize the receptor dimers, we carried out a thermodynamic study of these domains expressed as C-terminal fusion proteins with staphylococcal nuclease. Similar fusion constructs have been used successfully to investigate the oligomerization and association thermodynamics of a number of transmembrane sequences, including that of glycophorin A. Using SDS-PAGE analysis and sedimentation equilibrium analytical ultracentrifugation, we do not find strong, specific homo or hetero-interactions between the transmembrane domains of the erbB receptors in micellar solutions. Our results indicate that any preferential interactions between these domains in micellar solutions are extremely modest, of the order of 1 kcal mol(-1) or less. We applied a thermodynamic formalism to assess the effect of weakly interacting TM segments on the behavior of a covalently attached soluble domain. In the case of the ligand-bound EGFR ectodomain, we find that restriction of the ectodomain to the micellar phase by a hydrophobic TM, even in the absence of strong specific interactions, is largely sufficient to account for the previously reported increase in dimerization affinity.