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PubMed2001

Targeting and functional role of N-RAP, a nebulin-related LIM protein, during myofibril assembly in cultured chick cardiomyocytes.

Targeting and functional effects of N-RAP domains were studied by expression as GFP-tagged fusion proteins in cultured embryonic chick cardiomyocytes. GFP-tagged N-RAP was targeted to myofibril precursors, myofibril ends and cell contacts, expression patterns that are similar to endogenous N-RAP. The GFP-tagged N-RAP LIM domain (GFP-N-RAP-LIM) was targeted to the membrane in cells with myofibril precursors and cell-cell contacts. The GFP-tagged super repeats (N-RAP-SR) and the GFP-tagged domain normally found in between the super repeats and the LIM domain (N-RAP-IB) were each observed at sites of myofibril assembly, incorporating into myofibril precursors in a manner similar to full length N-RAP. However, unlike full-length N-RAP, N-RAP-SR and N-RAP-IB were also found in mature myofibrils, associating with the sarcomeric actin filaments and the Z-lines, respectively. N-RAP-IB was also colocalized with alpha-actinin at cell contacts. Each of the N-RAP constructs could inhibit the formation of mature myofibrils in cultured cardiomyocytes, with the effects of N-RAP-SR and N-RAP-IB depending on the time of transfection. The results show that each region of N-RAP is crucial for myofibril assembly. Combining the targeting and functional effects of N-RAP domains with information in the literature, we propose a new model for initiation of myofibrillogenesis.

Actinin↗

PubMed1994

Incorporation of Rap 1b into the platelet cytoskeleton is dependent on thrombin activation and extracellular calcium.

Rap 1b is a 22-kDa low molecular mass GTP-binding protein which is both a member of the Ras superfamily and a substrate for cAMP-dependent protein kinase. Recently, evidence has been presented to show that Rap 1b is incorporated into the detergent-extracted cytoskeleton of platelets during thrombin-induced activation. The aims of this study were to compare the incorporation of Rap 1b into the detergent-extracted cytoskeleton after activation with different agonists, to examine the role of extracellular calcium on the incorporation of Rap 1b into the cytoskeleton, to investigate the relationship between the association of Rap 1b and other proteins with the cytoskeleton, and to determine the effect of phosphorylation of Rap 1b incorporation into the cytoskeleton. Platelets were activated with thrombin, A23187, phorbol myristate acetate, ADP, epinephrine, and collagen in the presence and absence of calcium. The time dependence of Rap 1b incorporation into the detergent-extracted cytoskeleton was then measured. When platelets were activated by thrombin in the presence of extracellular calcium, conditions which permit aggregation, incorporation of Rap 1b into the detergent-extracted cytoskeleton was biphasic. Approximately 20% of the total cellular Rap 1b incorporated into the cytoskeleton within seconds and was followed by a slower second phase of incorporation. In contrast, when platelets were activated by thrombin in the absence of calcium, conditions which inhibit aggregation, or by the other agents in the presence or absence of calcium, only the initial phase of Rap 1b incorporation into the cytoskeleton was measured. The incorporation of Rap 1b paralleled the incorporation of membrane glycoproteins (GP) IIb/IIIa and PECAM-1, but not the incorporation of pp60c-src. The GTPase-activating protein for Ras (Ras-GAP) did not associate with the detergent-extracted cytoskeleton. Two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis of the total cellular and cytoskeletal Rap 1b showed that unphosphorylated as well as phosphorylated isoforms of Rap 1b were incorporated into the cytoskeleton in the same molar ratio as was present in the intact cell. Furthermore, the rates of incorporation of phosphorylated and unphosphorylated Rap 1b into the cytoskeleton were similar. These experiments show that Rap 1b can regulate events that take place within seconds after activation, such as the initial formation of the cytoskeleton, as well as longer term changes in the cytoskeleton that occur in response to thrombin-induced aggregation. Furthermore, phosphorylation could modulate the (unknown) functions of Rap 1b as a component of the cytoskeleton.

Blood Platelets↗

PubMed1999

Molecular interactions of N-RAP, a nebulin-related protein of striated muscle myotendon junctions and intercalated disks.

N-RAP is a recently discovered muscle-specific protein that is concentrated at the myotendon junctions in skeletal muscle and at the intercalated disks in cardiac muscle. The C-terminal half of N-RAP contains a region with sequence homology to nebulin, while a LIM domain is found at its N-terminus. N-RAP is hypothesized to perform an anchoring function, linking the terminal actin filaments of myofibrils to protein complexes located beneath the sarcolemma. We used a solid-phase assay to screen myofibrillar and junctional proteins for binding to several recombinant fragments of N-RAP, including the nebulin-like super repeat region (N-RAP-SR), the N-terminal half including the LIM domain (N-RAP-NH), and the region of N-RAP between the super repeat region and the LIM domain (N-RAP-IB). Actin is the only myofibrillar protein tested that exhibits specific binding to N-RAP, with high-affinity binding to N-RAP super repeats, and 10-fold weaker binding to N-RAP-IB. In contrast, myosin, isolated myosin heads, tropomyosin, and troponin exhibited no specific interaction with N-RAP domains. A recombinant fragment corresponding to the C-terminal one-fourth of vinculin also binds specifically to N-RAP super repeats, while no specific N-RAP binding activity was observed for other regions of the vinculin molecule. Finally, talin binds with high affinity to the LIM domain of N-RAP. These results support our hypothesis that N-RAP is part of a complex of proteins that anchors the terminal actin filaments of the myofibril to the membrane, and functions in transmitting tension from the myofibrils to the extracellular matrix.

Actins↗

PubMed1998

Sequence and functional analysis of the intergenic regions separating babesial rhoptry-associated protein-1 (rap-1) genes.

The rhoptry-associated protein 1 (RAP-1) expressed by all babesial parasites is encoded by tandemly arranged genes separated by discrete intergenic (IG) regions. We hypothesize that these IG regions regulate rap-1 gene expression. In Babesia bovis two identical rap-1 gene copies are separated by a 1.0-kb noncoding region which is also exactly conserved 5' to the rap-1 gene 1. In contrast, the complex B. bigemina rap-1 locus contains at least 5 polymorphic rap-1a genes separated by uncharacterized 3.38-kb regions. A genomic clone encoding the 3' sequence of rap-1 gene copy 1, the 1 kb IG region, and the 5' sequence of gene copy 2 was obtained by PCR amplification of DNA from the Mo7 biological clone of B. bovis and sequenced. This was follow by amplification and sequence analysis of the 3.38-kb region separating two B. bigemina rap-1a genes, revealing the presence of two different IG regions denominated IG-1 (0.7 kb) and IG-2 (1.3 kb), flanking a newly identified rap-1b orf. Sequence analysis and comparison among babesial rap-1 IG regions from B. bovis, B. bigemina, B. canis, and B. ovis revealed conservation of at least three putative regulatory boxes consistently positioned 5' of the start of the rap-1 orfs. To determine whether rap-1 IG regions contained a functional promoter, the entire 1-kb IG region from B. bovis was cloned into pCAT, a promoterless plasmid containing the cat gene. The IG region in the 5' --> 3' orientation strongly promoted transcription in vitro by homologous B. bovis RNA polymerases. The presence of conserved regions 5' to each rap-1 gene copy and among other babesial rap-1 IG regions and the in vitro promoter function in the 5' --> 3' orientation support a role for the IG region in rap-1 gene regulation.

Animals↗

PubMed2003

Genomic organization, alternative splicing, and expression of human and mouse N-RAP, a nebulin-related LIM protein of striated muscle.

Linkage analysis identifies 10q24-26 as a disease locus for dilated cardiomyopathy (DCM), a region including the N-RAP gene. N-RAP is a nebulin-like LIM protein that may mediate force transmission and myofibril assembly in cardiomyocytes. We describe the sequence, genomic structure, and expression of human N-RAP, as well as an initial screen to determine whether N-RAP mutations cause cardiomyopathy. Human expressed sequence tag databases were searched with the published 3,528-bp mouse N-RAP open reading frame (ORF). Putative cDNA sequences were interrogated by direct sequencing from cardiac and skeletal muscle RNA. We identified two human N-RAP isoforms with ORFs of 5,085 bp (isoform C) and 5,190 bp (isoform S), encoding products of 193-197 kDa. Genomic database searches localize N-RAP to human chromosome 10q25.3 and match isoforms C and S to 41 and 42 exons. Only isoform C is detected in human cardiac RNA; in skeletal muscle, approximately 10% is isoform C and approximately 90% is isoform S. We investigated apparent differences between human N-RAP cDNA and mouse sequences. Two mouse N-RAP isoforms with ORFs of 5,079 and 5,184 bp were identified with approximately 85% similarity to human isoforms; published mouse sequences include cloning artifacts truncating the ORF. Murine and human isoforms have similar gene structure, tissue specificity, and size. N-RAP is especially conserved within its nebulin-like and LIM domains. We expressed both N-RAP isoforms and the previously described truncated N-RAP in embryonic chick cardiomyocytes. All constructs targeted to myofibril precursors and the cell periphery, and inhibited myofibril assembly. Several human N-RAP polymorphisms were detected, but none were unique to cardiomyopathy patients. N-RAP is highly conserved and exclusively expressed in cardiac and skeletal muscle. Genetic abnormalities remain excellent candidate causes for cardiac and skeletal myopathies.

Alternative Splicing↗

PubMed1996

Oncogenic amino acid substitutions in the inhibitory rap-1A protein cause it to adopt a ras-p21-like conformation as computed using molecular dynamics.

rap-1A is a membrane-bound G-protein in the ras superfamily that, like the ras-p21 protein, is activated by binding GTP in place of GDP. When activated, however, this protein inhibits the action of ras-p21, which is to induce mitogenesis in cells A chimeric protein containing RAS-p21 residues 1-65 and rap-1A residues 66-184 becomes ras-p21-like in its activity. The critical changes in sequence that result in this transformation are G26N, 127H, E30D, K31E, and E45V. All of these substitutions occur in or around a critical effector domain of p21 that is involved in interacting with GTPase activating protein (GAP), raf-p74 protein and inositol-3-hydroxy kinase. Using molecular dynamics, we have computed the average low energy structures for each of the three proteins, ras-p21, rap-1A and mutant rap1A, called rap-M, that contains these critical amino acid substitutions. We find that rap-M more closely superimposes on ras-p21 (rms deviation 1.9 A) than on wild-type rap-1A (rms deviation 3.4 A). In particular, the amino terminal domains (residues 3-59) of both RAS-p21 and rap-M are superimposable while they deviate when the average structures of these two proteins are superimposed on that of wild-type rap-1A. We have identified Pro 34 as a critical residue which may determine if the protein transforms cells or inhibits cell transformation. In addition, we have found that ras-p21 and rap-M proteins are superimposable in the region 96-110 except at Asp 105. The 96-110 domain of ras-p21 has been found to be involved in the binding of this protein to the nuclear transcription protein, jun and its kinase, jun kinase, JNK. Both segments differ in structure from that of the rap-1A segment at Asp 108, implicating this residue as also being important in determining the activity of the protein. Overall, the oncogenic substitutions introduced into the rap-1A protein cause it to adopt a conformation that is very similar to that of ras-p21 rather than wild-type rap-1A.

Amino Acid Sequence↗

PubMed2001

Cytokine regulation of cartilage-derived retinoic acid-sensitive protein (CD-RAP) in primary articular chondrocytes: suppression by IL-1, bfGF, TGFbeta and stimulation by IGF-1.

Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a secreted protein identified in our laboratory by RT-PCR and differential display [U.H. Dietz, L.J. Sandell. Cloning of a retinoic acid-sensitive mDNA expressed in cartilage and during chondrogenesis. J. Biol. Chem. 271 (1996) 3311-3316]. It is synthesized by chondrocytes throughout development and down-regulated by retinoic acid in coordination with type II collagen gene expression. To further explore the regulation CD-RAP in primary articular chondrocytes, we examined effects of selected cytokines on CD-RAP gene expression compared to their effects on type II collagen expression. Northern blot analysis showed that expression of CD-RAP mRNA was suppressed by bFGF, IL-1beta and retinoic acid in coordination with type II collagen mRNA. TGF-beta decreased CD-RAP expression while increasing type II collagen mRNA whereas both mRNAs were up-regulated by IGF-1. In chondrocytes dedifferentiated with retinoic acid, IGF-1 induced re-expression of both CD-RAP and type II collagen mRNAs. The mechanism of stimulation of CD-RAP by IGF-1 was further investigated. An mRNA stability assay revealed that IGF-1 had no effect on CD-RAP or type II collagen mRNA half life, suggesting that the enhancement by IGF-1 is due to increased gene transcription. To study the transcriptional mechanism, we used the 5'-flanking region of the CD-RAP gene fused to a promoter-less reporter plasmid encoding luciferase. Deletion analysis of the CD-RAP promoter indicated that an IGF-1-responsive element is present between nucleotides -475 and -458. These data indicate that CD-RAP expression can be regulated by cytokines known to influence chondrocyte metabolism and that IGF-1 up-regulates CD-RAP gene expression through a transcriptional mechanism.

Animals↗

PubMed2001

Dominant thermodynamic role of the third independent receptor binding site in the receptor-associated protein RAP.

The 39 kDa receptor-associated protein (RAP) is a three-domain escort protein in the secretory pathway for several members of the low-density lipoprotein receptor (LDLR) family of endocytic receptors, including the LDLR-related protein (LRP). The minimal functional unit of LRP required for efficient binding to RAP is composed of complement-type repeat (CR)-domain pairs, located in clusters on the extracellular part of LRP. Here we investigate the binding of full-length RAP and isolated RAP domains 1-3 to an ubiquitin-fused CR-domain pair consisting of the fifth and sixth CR domains of LRP (U-CR56). As shown by isothermal titration calorimetric analysis of simple RAP domains as well as adjoined RAP domains, all three RAP domains bind to this CR-domain pair in a noncooperative way. The binding of U-CR56 to RAP domains 1 and 2 is (at room temperature) enthalpically driven with an entropy penalty (K(D) = 2.77 x 10(-6) M and 1.85 x 10(-5) M, respectively), whereas RAP domain 3 binds with a substantially lower enthalpy, but is favored due to a positive entropic contribution (K(D) = 1.71 x 10(-7) M). The heat capacity change for complex formation between RAP domain 1 and the CR-domain pair is -1.65 kJ K(-1) mol(-1). There is an indication of a conformational change in RAP domain 3 upon binding in the surface plasmon resonance analysis of the interaction. The different mechanisms of binding to RAP domains 1 and 3 are further substantiated by the different effects on binding of mutations of the Asp and Trp residues in the LRP CR5 or CR6 domains, which are important for the recognition of several ligands.

Binding Sites↗

PubMed1998

Deficient post-translational processing of Rap 1A in variant HL-60 cells.

Variant HL-60 cells resistant to differentiation induced by nitroprusside and cGMP analogs have normal guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity (J. Biol. Chem. 269, 32155-32161, 1994). We found decreased phosphorylation of a low molecular weight protein (pp23) in the variant cells and by co-migration on two-dimensional polyacrylamide gels, phosphopeptide mapping, immunoprecipitation and immunoblotting, we showed that pp23 was one of three post-translationally modified forms of Rap 1A expressed in HL-60 cells. Using an in vitro transcription/translation system, we studied each of the posttranslational processing steps of Rap 1A and we showed that pp23 represented fully processed Rap 1A. By immunoprecipitation, immunoblotting and 35S-methionine/cysteine incorporation, we showed that the variant cells were deficient in pp23, and thus in fully processed Rap 1A, but that these cells did express normal amounts of completely unprocessed Rap 1A and geranylgeranylated Rap 1A; the lack of Rap 1A processing beyond geranylgeranylation in the variant cells was not secondary to a change in Rap 1A's amino acid sequence. The variant cells had normal carboxyl methyltransferase activity suggesting they are deficient in proteolytic cleavage of Rap 1A. The deficient post-translational processing of Rap 1A had no effect on Rap 1A's subcellular distribution and we found no evidence for altered post-translational processing of H-Ras.

Cyclic GMP↗

PubMed1998

Analysis of inhibitory epitopes in the Plasmodium falciparum rhoptry protein RAP-1 including identification of a second inhibitory epitope.

Immune responses to Plasmodium falciparum rhoptry-associated protein 1 (RAP-1), RAP-2, and RAP-3 appear to contribute to protection against infection by this human malarial parasite. This conclusion is suggested by results of monkey immunization trials and of cell culture studies showing antibody-dependent inhibition of erythrocyte invasion. In the present study, splenectomized owl monkeys were infected with P. falciparum in order to monitor anti-RAP-1 antibody production as antiparasite immunity developed. The monkeys responded to a primary infection with the production of antibodies to a fragment of RAP-1 containing amino acids 1 to 294 (RAP-1(1-294)). After drug cure and reinfection, the monkeys had a prolonged prepatent period, indicating they had already developed partial immunity to the parasite. Sera from these animals showed major increases in anti-RAP-1(1-294) antibodies. In contrast, only low levels of antibodies to inhibitory B-cell epitope 1 (iB-1), an inhibitory epitope in RAP-1(1-294) with the sequence N200TLTPLEELYPT211, was observed after the initial parasite infection, and the anti-iB-1 antibodies were not readily boosted upon reinfection. These results suggest that iB-1 is an immunogenic but not immunodominant epitope and that anti-iB-1 antibodies do not substantially contribute to early stages of naturally acquired immunity in the owl monkey model. To identify additional epitopes bound by inhibitory antibodies, mouse monoclonal antibodies were produced with a recombinant fusion protein containing RAP-1(1-294). Monoclonal antibody 1D6 inhibited parasite invasion of erythrocytes in vitro. 1D6 did not bind peptide iB-1 but rather bound a second inhibitory epitope called iB-2. iB-2, like iB-1, is found near the amino terminus of p67, a RAP-1 processing product thought to be involved in merozoite invasion of erythrocytes. Since anti-iB-1 antibodies were not readily produced during parasite infection, it may be desirable to direct antibody responses to particular epitopes in RAP-1, such as iB-1 and iB-2.

Amino Acid Sequence↗

PubMed2004

Efficient transfer of receptor-associated protein (RAP) across the blood-brain barrier.

We have sought to identify a high-capacity transport system that mediates transcytosis of proteins from the blood to the brain. The 39 kDa receptor-associated protein (RAP) functions as a specialized endoplasmic reticulum chaperone assisting in the folding and trafficking of members of the low-density lipoprotein (LDL) receptor family. RAP efficiently binds to these receptors and antagonizes binding of other ligands. Previous studies have shown that two large members of the LDL receptor family, LDL receptor-related protein 1 (LRP1) and LDL receptor-related protein 2 (LRP2 or megalin), possess the ability to mediate transcytosis of ligands across the brain capillary endothelium. Here, we tested whether blood-borne RAP crosses the blood-brain barrier (BBB) by LRP1- or megalin-mediated transport by studying the pharmacokinetics of [125I]-RAP transport into the brain in intact mice and across cell monolayers in vitro. Our results show that [125I]-RAP is relatively stable in blood for 30 minutes and has a mean influx constant of 0.62+/-0.08 microl/g-minute from blood to brain. In situ brain perfusion in blood-free buffer shows that transport of [125I]-RAP across the BBB is a saturable process. Capillary depletion of brain homogenates indicates that 70% of [125I]-RAP is localized in the parenchyma rather than in the vasculature of the brain. Results of transport in stably transfected MDCK cells are consistent with the hypothesis that megalin mediates most of the apical-to-basolateral transport across polarized epithelial cells. The inhibition of [125I]-RAP influx by excess RAP and the involvement of megalin indicate the presence of a saturable transport system at the BBB. The higher permeability of RAP compared with that of melanotransferrin and transferrin show that the LRP receptor is a high capacity transport system. These studies suggest that RAP may provide a novel means of protein-based drug delivery to the brain.

Animals↗

PubMed1991

Inhibition of ras-induced germinal vesicle breakdown in Xenopus oocytes by rap-1B.

A cDNA clone (Krev-1) has recently been identified that possesses the ability to reverse the transformed phenotype when introduced into a K-ras-transformed NIH/3T3 cell line. The Krev-1 protein, also known as rap-1A, was found to share 50% homology with the ras proteins. The rap-1A protein has also been shown to block the interaction of ras with its GTPase activating protein in vitro, leading to speculation regarding its role in vivo. A closely related protein, rap-1B, has also been identified in platelets, human erythroleukemia cells, neutrophils, and aortic smooth muscle cells. Unlike rap-1A, rap-1B has been shown to be phosphorylated in platelets. Given the high degree of similarity between the amino acid sequences of rap-1A and rap-1B, we sought to investigate the effect of microinjected rap-1B on H-ras(Val12)-induced germinal vesicle breakdown in Xenopus laevis oocytes. In this assay system, equimolar concentrations of rap-1B were found to block germinal vesicle breakdown triggered by the oncogenic ras protein. However, in the presence of IGF-1, this inhibition was not observed. Moreover, rap-1B is readily phosphorylated in the oocytes.

Animals↗

PubMed2000

The receptor-associated protein (RAP) interacts with several resident proteins of the endoplasmic reticulum including a glycoprotein related to actin.

The receptor-associated protein (RAP) is a chaperone found primarily in the endoplasmic reticulum (ER) that plays a necessary role in the folding and exocytic trafficking of members of the LDL receptor gene family including megalin and the LDL receptor-related protein (LRP). Recently, RAP has been shown to interact with a growing number of proteins including several that are unrelated to the LDL receptor family as well as new members of this rapidly expanding family. Based on these observations, we have applied chemical crosslinking procedures to identify additional novel RAP-interacting proteins, and thereby better characterize the scope of RAP's ER-related function. In this study, we have identified eight proteins with molecular weights of 32, 35, 46, 55, 70, 95, 170, and 200 kDa that interact with endogenous RAP. These proteins were found to associate with RAP in multiple cell types from different species, suggesting that their expression and interactions with RAP are ubiquitous. Results of pulse-chase experiments show that most of the proteins remain sensitive to endoglycosidase-H digestion, and also remain stably associated with RAP over an extended period, suggesting that they are ER resident proteins. All of the RAP-associated proteins appear to be largely soluble as they partition into the aqueous phase following TX-114 detergent extraction. Sequence analysis and immunoblotting of the 46-kDa RAP-associated glycoprotein (gp46) shows that it is structurally and immunologically related to actin. If gp46 is also functionally related to actin as an intracellular structural protein, it may represent a novel component of the putative ER matrix.

Actins↗

PubMed2003

Characterization of RAP, a quorum sensing activator of Staphylococcus aureus.

Staphylococcus aureus are Gram-positive bacteria and cause diverse serious diseases in humans and animals through the production of toxins. The production of toxins is regulated by quorum sensing mechanisms, where proteins such as RNAIII activating protein (RAP) are secreted by the bacteria and induce virulence. Antibodies to RAP have been shown to protect mice from infection, but the molecular structure of RAP was not known and hindered vaccine development. To characterize RAP, recombinant protein was made and tested for its ability to induce genes important for pathogenesis (agr). In addition, monoclonal antibodies were produced to identify its cellular localization. Results shown here indicate that RAP is a 277-aa protein that is an ortholog of the ribosomal protein L2. Like the native molecule, recombinant RAP activates the production of RNAIII (encoded by agr). Using RAP specific monoclonal antibodies we demonstrate that RAP is continuously secreted and while RAP is expressed also in other bacteria (like Staphylococcus epidermidis, Staphylococcus xylosus and Escherichia coli), it is secreted to the culture medium only by S. aureus. Our results show that the ribosomal protein L2 has an extraribosomal function and that when secreted RAP acts as an autoinducer of virulence to regulate S. aureus pathogenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Induction of CD-RAP mRNA during periosteal chondrogenesis.

Induction of chondrogenesis and maintenance of the chondrocyte phenotype are critical events for autologous periosteal transplantation, which is a viable approach for cartilage repair. Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a recently discovered protein that is mainly produced in cartilage. During development, CD-RAP expression starts at the beginning of chondrogenesis and continues throughout cartilage maturation. In order to investigate the involvement of CD-RAP during periosteal chondrogenesis we have determined the nucleotide sequence of the rabbit CD-RAP mRNA and utilized this information to evaluate the temporal and spatial expression pattern of CD-RAP at the mRNA level during chondrogenesis. When the periosteal explants were cultured under chondrogenic conditions, the expression of CD-RAP was induced, as shown by a 40-fold increase in CD-RAP mRNA between days 7 and 10. The temporal expression pattern of CD-RAP closely mimicked that of collagen type IIB mRNA. Also, the CD-RAP mRNA was localized to the matrix forming chondrocytes in the cambium layer of the periosteum by in situ hybridization as indicated by colocalization with collagen type II mRNA and positive safranin O staining. These data suggest a regulatory role of CD-RAP in periosteal chondrogenesis, which is potentially important for both cartilage repair and fracture healing via callus formation.

Amino Acid Sequence↗

PubMed2001

Ultrastructural and biochemical localization of N-RAP at the interface between myofibrils and intercalated disks in the mouse heart.

N-RAP is a recently discovered muscle-specific protein found at cardiac intercalated disks. Double immunogold labeling of mouse cardiac muscle reveals that vinculin is located immediately adjacent to the fascia adherens region of the intercalated disk membrane, while N-RAP extends approximately 100 nm further toward the interior of the cell. We partially purified cardiac intercalated disks using low- and high-salt extractions followed by density gradient centrifugation. Immunoblots show that this preparation is highly enriched in desmin and junctional proteins, including N-RAP, talin, vinculin, beta1-integrin, N-cadherin, and connexin 43. Electron microscopy and immunolabeling demonstrate that N-RAP and vinculin are associated with the large fragments of intercalated disks that are present in this preparation, which also contains numerous membrane vesicles. Detergent treatment of the partially purified intercalated disks removed the membrane vesicles and extracted vinculin and beta1-integrin. Further separation on a sucrose gradient removed residual actin and myosin and yielded a fraction morphologically similar to fasciae adherentes that was highly enriched in N-RAP, N-cadherin, connexin 43, talin, desmin, and alpha-actinin. The finding that N-RAP copurifies with detergent-extracted intercalated disk fragments even though beta-integrin and vinculin have been completely removed suggests that N-RAP association with the adherens junction region is mediated by the cadherin system. Consistent with this hypothesis, we found that recombinant N-RAP fragments bind alpha-actinin in a gel overlay assay. In addition, immunofluorescence shows that N-RAP remains bound at the ends of isolated, detergent-treated cardiac myofibrils. These results demonstrate that N-RAP remains tightly bound to myofibrils and fasciae adherentes during biochemical purification and may be a key constituent in the mechanical link between these two structures.

Animals↗

PubMed1994

Functional domains of the receptor-associated protein (RAP).

The receptor-associated protein (RAP) specifically associates with gp330 and the low density lipoprotein (LDL) receptor-related protein (LRP), the two newest members of the LDL receptor gene family. Results obtained by ligand blotting, affinity chromatography, and density-gradient sedimentation demonstrate that RAP binds to both receptors with high affinity and that the binding is Ca2+ dependent. RAP also binds heparin and is identical to a mouse heparin binding protein (HBP-44) identified in a teratocarcinoma cell line (F9). While biochemical studies have shown that RAP is present on the cell surface and is an effective inhibitor of ligand binding to gp330 and LRP, immunocytochemical findings indicate that RAP is most abundant in the endoplasmic reticulum lumen and may function in receptor folding and/or trafficking. To facilitate the characterization of RAP's function(s) we have mapped its gp330 and heparin binding sites by performing direct binding studies on fusion proteins representing overlapping domains of RAP. gp330 was found to bind to two separate sites on RAP--i.e., between amino acids 85-148 and 178-248. Binding studies with radiolabeled heparin indicate that the heparin binding site is between amino acids 261 and 323, which is consistent with our previously proposed site (residues 287-306) based on the amphipathic nature of the C terminus of RAP. These data demonstrate that the gp330 and heparin binding sites and the Heymann nephritis pathogenic epitope (amino acids 1-86) demonstrated earlier are represented by distinct domains of the RAP polypeptide.

Binding Sites↗

PubMed1996

Educating patients at home. Community Health Rap.

OBJECTIVES: The authors analyzed the impact of home health education by studying the impact of a computer service called Community Health Rap. When patients call this service, the computer records their questions and alerts an expert who records a response. Subsequently, the computer alerts the patient that the question has been addressed. METHODS: Subjects included a group of 82 pregnant women who had used cocaine during or 1 month before pregnancy (as reported by the woman) and a group of residents of zip code areas with the lowest income in Cleveland. From the drug-using pregnant women, we collected data regarding satisfaction with Community Health Rap, usage of Community Health Rap per month, self-reported health status (using the General Health Survey), and the extent of drug use (using the Addiction Severity Index). Trained coders also classified the nature of questions posed to the Community Health Rap by either the pregnant women who abuse drugs or the members of target households. Among the pregnant women who abuse drugs, we compared the differences between those who used the service and those who did not. To control for baseline differences between the two groups, analysis of co-variance was used with exit values as the dependent variables, the baseline values as the co-variates, and participation in the Community Health Rap as the independent variable. RESULTS: Almost half (45%) of poor, undereducated subjects who lived in inner urban areas used the computer service. Content analysis of Community Health Rap messages revealed that subjects had many questions that were of a social nature (regarding sex, relationships, etc), in addition to medical questions. Analysis showed that poor health status, more frequent drug use, lower education, and age did not affect regular use of Community Health Rap service. No health outcomes or utilization of treatment were associated with regular use of Community Health Rap. One exception, however, was that regular users of Community Health Rap reported slower improvement of their pain than those who did not use Community Health Rap. CONCLUSIONS: These data suggest that expansion of information services to households will not leave the poor and the undereducated population groups "behind." They will use computer services, though such services may not have an impact on their health status or cost of care.

Adult↗