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PubMed2005

Defective thyroglobulin storage in LDL receptor-associated protein-deficient mice.

The molecular chaperone receptor-associated protein (RAP) is required for biosynthesis of megalin, an endocytic receptor for follicular thyroglobulin (Tg), the thyroid hormone precursor. RAP also binds to Tg itself, suggesting that it may affect Tg trafficking in various manners. To elucidate RAP function, we have studied the thyroid phenotype in RAP-knockout (RAP-KO) mice and found a reduction of Tg aggregates into thyroid follicles. Serum Tg levels were significantly increased compared with those of wild-type (WT) mice, suggesting a directional alteration of Tg secretion. In spite of these abnormalities, hormone secretion was maintained as indicated by normal serum thyroxine levels. Because Tg in thyroid extracts from RAP-KO mice contained thyroxine residues as in WT mice, we concluded that in RAP-KO mice, follicular Tg, although reduced, was nevertheless sufficient to provide normal hormone secretion. Serum TSH was increased in RAP-KO mice, and although no thyroid enlargement was observed, some histological features resembling early goiter were present. Megalin was decreased in RAP-KO mice, but this did not affect thyroid function, probably because of the concomitant reduction of follicular Tg. In conclusion, RAP is required for the establishment of Tg reservoirs, but its absence does not affect hormone secretion.

Animals↗

PubMed1987

Ventricular performance related to transmural filling pressure in clinical cardiac tamponade.

In clinical cardiac tamponade, open-catheter intrapericardial pressure (IPP) may be used to estimate left ventricular transmural filling pressure (TMFP). However, it has been suggested recently that right atrial pressure (RAP) is superior to IPP in assessing true extracardiac pressure during pericardial drainage. In 10 patients with subacute cardiac tamponade, pulmonary wedge pressure (PWP), RAP, and IPP were measured along with indexes of systolic function. To test the relative merits of IPP and RAP in assessing true pericardial pressure, three TMFP estimates were analyzed: TMFP1 = (PWP - IPP); TMFP2 = (PWP - 1/3 RAP - 2/3 IPP); and TMFP3 = (PWP - RAP). An accurate TMFP presumably should increase during pericardiocentesis and correlate with left ventricular stroke work. In addition, to test the role of preload variation in pulsus paradoxus, respiratory variation in TMFP was analyzed. In the initial tamponade state, RAP and IPP were essentially equal, so all three TMFP estimates gave equivalent results. For instance, TMFP1 averaged 4 +/- 2 mm Hg but fell to 0.2 +/- 1.3 mm Hg during inspiration (p less than .001 vs expiration) and showed beat-by-beat correlation with pulse arterial pressure. After intermediate pericardiocentesis (280 +/- 160 ml), the IPP of 6 +/- 3 mm Hg fell significantly below the RAP of 10 +/- 3 mm Hg (p less than .001), but with a 570 +/- 320 ml residual effusion suggesting continued IPP measurement accuracy. By complete pericardiocentesis (810 +/- 430 ml) there was a significant increase in TMFP1 to 8 +/- 4 mm Hg (p less than .05 vs tamponade) but not in the TMFP3 of 1 +/- 3 mm Hg. Encompassing tamponade and pericardiocentesis data, left ventricular stroke work index showed positive correlation with TMFP1 (r = .59) and TMFP2 (r = .52) but not with TMFP3. Thus cardiac tamponade often may be diagnosed with a TMFP averaging well above zero, and diastolic equalization of PWP, RAP, and IPP may be a predominantly inspiratory finding ("inspiratory tracking"). This supports the role of preload variation in the genesis of pulsus paradoxus. On the other hand, true pericardial pressure may fall substantially below RAP in the course of pericardial drainage. This may be reconciled with the concept that normal pericardial pressure nearly equals RAP by hypothesizing an increased pericardial capacity in subacute tamponade so that pericardiocentesis produces a state analogous to removal of normal pericardial constraint.

Cardiac Tamponade↗

PubMed1998

Calcium/calmodulin kinase inhibitors and immunosuppressant macrolides rapamycin and FK506 inhibit progestin- and glucocorticosteroid receptor-mediated transcription in human breast cancer T47D cells.

The effects of immunosuppressants and inhibitors of specific calcium/calmodulin kinase (CaMK) of types II and IV on progestin/glucocorticosteroid-induced transcription were studied in two human stably transfected breast cancer T47D cell lines. The lines contain the chloramphenicol acetyl transferase (CAT) gene under control either of the mouse mammary tumor virus promoter (T47D-MMTV-CAT), or the minimal promoter containing five glucocorticosteroid/progestin hormone response elements [T47D-(GRE)5-CAT]. Progestin- and triamcinolone acetonide (TA)-induced CAT gene expression was inhibited in a dose-dependent manner in both lines by preincubation with rapamycin (Rap) and, to a lesser extent, with FK506, but not with cyclosporin A. CaMK II and/or IV inhibitors KN62 and KN93 also inhibited progestin- and TA-stimulated transcription in both lines. None of these drugs had any effect on basal transcription. The antagonist RU486 inhibited all the effects of both progestin and TA, suggesting that progesterone receptor (PR)-, as well as glucocorticosteroid receptor (GR)- mediated transactivation are targets of immunosuppressants and CaMKs in T47D cells. Indeed, Northern analysis showed that Rap, KN62, and, to a lesser degree, FK506 inhibited progestin stimulation of Cyclin D1 mRNA levels, but not those of the non-steroid-regulated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Addition of Rap or KN62 after exposure of cells to progesterone agonist Org 2058 had no effect on induction of CAT activity. Taken together, these data indicate that Rap and FK506, as well as CaMK inhibitors, inhibit steroid-induced activities of exogenous, as well as of some endogenous, steroid receptor-regulated genes by a mechanism preceding hormone-induced receptor activation. Rap appeared to stabilize a 9S form of [3H]Org 2058-PR complexes isolated from T47D (GRE)5CAT cell nuclei. By contrast, the progesterone receptor (PR) was isolated from cells treated with KN62 as a 5S entity, undistinguishable from the 5S PR species extracted from cells treated with progestin only. The nuclear 9S-[3H]Org2058-PR resulting from cells exposed to Rap, contained, in addition to the heat shock proteins of 90 kDa and 70 kDa (hsp90 and hsp70), the FK506-binding immunophilin FKBP52 but not FKBP51, although the latter was part of unliganded PR heterocomplex associated with hsp90. These results suggest that Rap and KN62 act upon the PR by distinct mechanisms, with only Rap impeding progestin-induced PR transformation. FKBP51 appeared to dissociate from the receptor heterocomplex, but not from hsp90, after hormone binding to PR in vitro and in vivo, whether in the presence or not of Rap and KN62. Immunoprecipitation experiments distinguished two PR- and glucocorticosteroid (GR)-associated molecular chaperone complexes, containing hsp90 and hsp70 and FKBP52 or FKBP51. Another complex identified in T47D cytosol contained hsp90 and the cyclosporin A-binding cyclophilin of 40 kDa, CYP40, but not hsp70, PR, or GR. These observations support the concept that FKBP51 and FKBP52 can act as regulators of Rap and FK506 activity upon PR and GR-mediated transcription, a mechanism that could be also regulated by type II and/or type IV CaMKs.

Animals↗

PubMed1992

The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, alpha 2-macroglobulin receptor and glycoprotein 330.

The 39-kDa receptor-associated protein (RAP) binds to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and inhibits binding of ligands to this receptor. The in vivo function of RAP may be to regulate ligand binding and/or assist in the correct biosynthetic processing or trafficking of the alpha 2MR/LRP. Here we show that RAP binds another putative receptor, the kidney glycoprotein 330 (gp330). Gp330 is a high molecular weight glycoprotein that is structurally similar to both the alpha 2MR/LRP and low density lipoprotein receptor. The ability of RAP to bind to gp330 was demonstrated by ligand blotting and solid phase binding assays, which showed that RAP binds to gp330 with high affinity (Kd = 8 nM). Exploiting the interaction of gp330 and RAP, we purified gp330 by affinity chromatography with a column of RAP coupled to Sepharose. Gp330 preparations obtained by this procedure were notably more homogeneous than those obtained by conventional methods. Immunocytochemical staining of human kidney sections localized RAP to the brush-border epithelium of proximal tubules. The fact that gp330 is also primarily expressed by proximal tubule epithelial cells strengthens the likelihood that the interaction between gp330 and RAP occurs in vivo. The functional significance of RAP binding to gp330 may be to antagonize ligand binding as has been demonstrated for the alpha 2MR/LRP or to assist in the biosynthetic processing and/or trafficking of this receptor.

Carrier Proteins↗

PubMed1990

[Recurrent abdominal pain in schoolchildren 9-12 years of age].

The frequency of recurrent abdominal pain (RAP) was investigated in a material of 664 Danish schoolchildren. 15% of the children aged 9-12 years had RAP, defined as at least three episodes of abdominal pain during a period of three months with pain of an intensity which affected the behavior of the child. In general, there was no significant difference in the frequency of RAP in boys and girls in the material, however, a preponderance of girls with RAP after the age of ten years was found. The investigation showed that symptoms such as headache, limb pains and diarrhoea were experienced more frequently among children with RAP. In general, RAP occurred more frequently among children in families with high frequencies of psychosomatic symptoms. 40% of all the children questioned lived in broken homes. A tendency to increased frequency of RAP was found among these children. Children who lived in a garden city had essentially lower frequencies of RAP than children from other areas. There was an evident preponderance of children with RAP in families who lived in flats. There was no significant difference in the school and occupational education of the parents and the frequency of RAP.

Abdominal Pain↗

PubMed1994

Relationship between multiple biologic effects of rapamycin and the inhibition of pp70S6 protein kinase activity. Analysis in mutant clones of a T cell lymphoma.

Rapamycin (RAP) inhibits several biologic responses in the YAC-1 T cell lymphoma, including the serum-driven proliferation and cyclin A mRNA expression, the induction of Ly-6E Ag expression by IFN, and the induction of IFN-gamma production by IL-1. RAP also suppresses the enzymatic activity of the 70 kDa S6 protein kinase (pp70s6k). To define the mechanistic relationship between these multiple effects of RAP, we have generated stable somatic mutants with altered sensitivities to this drug. A first series of mutants, represented by the R19, 4R16, and 10R13 clones, showed markedly reduced sensitivity to the inhibitory effect of RAP on all biologic responses tested and on pp70s6k activity. Two other mutant types, R103 and R125, were both highly sensitive to RAP-mediated suppression of proliferation, of IL-1-induced IFN-gamma production, and of pp70s6k activity but differed in their Ly-6E response. This response was not affected by RAP in the R125 clone and was enhanced in the R103 clone. Therefore, the inhibitory effects of RAP on proliferation and IL-1-mediated IFN-gamma induction both appear associated with the inhibition of pp70s6k activity, whereas the modulation of Ly-6E induction is independent from the latter. Moreover, the cellular binding of [3H]dihydro-FK-506 was found to be blocked by RAP in all mutant types to the same extent as in wild-type YAC-1 cells, suggesting that the altered sensitivity to the effects of RAP in these mutants is not due to an inability of the drug to enter the cells or to interact with FKBP. Further biochemical characterization of the mutant cells described here is expected to help clarify the mechanisms of RAP action.

Animals↗

PubMed1993

Failure of rapamycin to block proliferation once resting cells have entered the cell cycle despite inactivation of p70 S6 kinase.

Rapamycin (RAP) has recently been shown to inhibit the phosphorylation and activity of p70 S6 kinase (p70s6k). In interleukin (IL)-2-induced cell activation of the human IL-2-dependent T-cell line, Kit225, RAP inhibited p70s6k phosphorylation and activation, but not the activation of MAP kinase, p90 S6 kinase (p90rsk), early tyrosine kinases, or the transcription of the c-fos and c-myc genes. Cell cycle progression induced by IL-2 was arrested by RAP prior to p110Rb phosphorylation and the major increase in total RNA synthesis, both of which were initiated around 6 h after addition of IL-2 and 9 h before the beginning of DNA synthesis. Interestingly, RAP could not inhibit DNA synthesis if addition of the drug was delayed for 6 h after addition of IL-2, despite the fact that even at this time, RAP rapidly induced the accumulation of the dephosphorylated form of p70s6k and that p70s6k was inactivated within 1 h of RAP addition. Furthermore, when RAP was added to continuously growing Kit225 cells, cell proliferation was maintained for at least two additional cell cycles, in the absence of apparent p70s6k activity. These results indicate that 1) among the earliest detectable signals after IL-2 treatment, RAP selectively inhibits p70s6k activation, 2) RAP inactivates p70s6k regardless of the stage of the cell cycle in which the drug is added, 3) RAP blocks resting T-cells from entering the cell cycle, but does not directly arrest cell cycle progression once cells have entered the cycle, and 4) inactivation of p70s6k does not cause immediate arrest of cell cycle progression once cells have entered the cycle.

Amino Acid Sequence↗

PubMed1991

Purification of a plasma membrane-associated GTPase-activating protein specific for rap1/Krev-1 from HL60 cells.

rap1/Krev-1 is a p21ras-related GTP-binding protein that has been implicated in the reversion of the ras-transformed cell phenotype. We have identified a GTPase-activating protein (GAP) specific for rap in plasma membranes isolated from differentiated HL60 cells. The rap GAP activity remained quantitatively associated with the membrane following washes with buffered 1 M LiCl containing 20 mM EDTA but was solubilized with the detergents Nonidet P-40 and deoxycholate. On the basis of size-exclusion chromatography, the membrane-associated rap GAP (rap GAPm) appeared distinct from the rap GAP detected in the cytosolic fraction from HL60 cells. The molecular sizes of the membrane and cytosolic forms were estimated to be 36 and 54 A, respectively. rap GAPm was solubilized and purified to near homogeneity by successive column chromatographies in the presence of detergent. The rap GAPm activity corresponded to a single polypeptide that migrated with a molecular mass of approximately 88 kDa on SDS/polyacrylamide gels. The purified rap GAPm was inactive toward the GTP-bound forms of p21ras, rho, G25K, and rac-1 and did not stimulate dissociation of guanine nucleotide from rap.

Amino Acid Sequence↗

PubMed1995

Babesia bigemina: identification of B cell epitopes associated with parasitized erythrocytes.

Rhoptries are involved in host cell invasion and rhoptry polypeptides, including the Babesia bigemina rhoptry-associated protein-1 (RAP-1), are targets for protective immune responses. Polyclonal antisera produced against isolated rhoptries is directed predominantly against RAP-1 and reacts with both the merozoite and the membrane of parasitized erythrocytes. To determine whether these B cell epitopes associated with the parasitized erythrocyte are derived from RAP-1 or, alternatively, from previously undetected merozoite polypeptides, monoclonal antibodies (mAbs) were generated from mice immunized with rhoptries isolated from the JG-29 clone of the Mexico strain. The anti-RAP-1 mAbs bound only merozoites in a punctate immunofluorescence pattern. A second group of four mAbs, none of which were reactive with RAP-1, bound the parasitized erythrocyte. Two of these latter mAbs, 64/44.17.3 and 64/05.7.2, reacted only with parasitized erythrocytes that had been permeabilized. MAb 64/44.17.3 bound a 54-kDa merozoite polypeptide while 64/05.7.2 bound a > or = 225-kDa merozoite polypeptide. MAbs 64/32.8.5 and 64/38.5.3 recognized epitopes on 17.5- and 76-kDa polypeptides exposed on the external surface of intact parasitized erythrocytes. The results indicate that the identified RAP-1 epitopes are not associated with the erythrocyte cytoskeleton or membrane and that anti-RAP-1 immunity is most likely generated against the free merozoite. All new mAbs reacted with every B. bigemina strain tested (Mexico, Puerto Rico, St. Croix, Texcoco, Jaboticabal). The conservation of RAP-1 epitopes among these strains supports the continued testing of RAP-1 as a vaccine component. In addition, the identification of epitopes expressed on the surface of erythrocytes infected with all five strains provides new candidate immunogens.

Animals↗

PubMed1992

Non invasive estimation of mean right atrial pressure utilizing the 2D-Echo transverse diameter of the left hepatic vein.

Aim of our study was to assess if the 2D-Echo transverse diameter of the left hepatic vein (LHV) obtained from the subcostal approach is correlated with invasive mean right atrial pressure (RAP). Thirty-two consecutive patients with acute myocardial infarction were studied by simultaneous 2D-Echo and right heart catheterization. LHV could be adequately imaged in 90% of patients. Mean RAP values at catheterization ranged from 0 to 15 mmHg (mean 6 +/- 3.5 mmHg). A correlation was found between mean RAP and LHV diameter calculated after inspiration (r = 0.81, SEE = 1.54 mm, slope = 0.71, p less than 0.001) as well as after expiration (r = 0.82, SEE = 1.32 mm, slope = 0.62, p less than 0.001) or during post-expiratory apnoea (r = 0.85, SEE = 1.3 mm, slope = 0.71, p less than 0.001). Moreover, percent increments of LHV diameter correlated well with percent changes of mean RAP during rapid infusion of 250-500 cc of saline in 4 patients (r = 0.98, p less than 0.001). Thus, the 2D-Echo LHV diameter is a feasible measurement that seems to correlate well with invasive mean RAP over a wide range of mean RAP values in patients with myocardial infarction; the changes in LHV diameter correlate well with the percent increase in central venous pressure obtained with saline infusion. Underestimation of invasive RAP occurs when mean RAP value exceeds 12 mmHg and can be explained by limited venous compliance. However these preliminary results suggest that this method may represent a valid alternative to obtain a non invasive estimate of mean RAP with 2D-Echocardiography.

Adult↗

PubMed1990

Plasma concentrations of atrial natriuretic peptide during physical exercise in patients with congestive heart failure.

Plasma concentrations of atrial natriuretic peptide (ANP) were measured in 25 patients with organic heart disease during physical exercise (baseline and maximum workload) in order to investigate if the responsiveness of stimulated release of ANP is still preserved in patients with heart failure and chronically elevated cardiac filling pressures. Since plasma concentrations of ANP are known to be positively correlated with mean right atrial pressures (RAP), the patients were divided into two groups according to their resting RAP; group I; those with normal RAP (less than or equal to 5 mmHg; n = 11); group II; those with elevated RAP (greater than 5 mmHg; n = 14). Under baseline conditions RAP (3.2 +/- 0.4 mmHg vs. 8.8 +/- 0.7 mmHg; p less than 0.01), pulmonary artery diastolic pressure (PADP; 9.5 +/- 0.9 mmHg vs. 17.9 +/- 1.8 pg/ml; p less than 0.01), and plasma ANP levels (128 +/- 19 pg/ml vs. 204 +/- 60 pg/ml; p less than 0.06) were significantly lower in group I than in group II. Both at rest and during maximum workload, plasma ANP concentrations were closely related to RAP, PADP, and mean pulmonary artery pressures in both groups. During exercise in all patients, RAP and PADP significantly increased, as well as plasma ANP concentrations. Similar increments in plasma ANP concentrations were accompanied by greater changes in RAP in group II than in group I. However, identical changes in PADP lead to identical increments in plasma ANP concentrations in both groups. In conclusion, the increments of plasma ANP concentrations during physical exercise were independent of the resting values of PADP, RAP, and plasma ANP concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1995

Gradient of pressure and time between proximal anal canal and high-pressure zone during internal anal sphincter relaxation. Its role in the fecal continence mechanism.

PURPOSE: The normal response to rectal distention is a relaxation of the proximal anal canal (PAC). We hypothesized that this mechanism would require a gradient of pressure and time to preserve continence. METHODS: Sixteen volunteers (10 male), mean age, 41.5 (range, 24-60) years, were studied using an eight port axial catheter with a compliant balloon at its tip. Relaxation was induced by a small volume of rectal distention (15-30 ml of air) and was recorded until recovery of resting anal pressure (RAP). Duration of relaxation was measured until recovery of RAP. Amplitude of relaxation was determined between RAP before rectal distention (RAP-BR) and pressure at the point of maximum relaxation (RAP-PMR). Gradient of pressure was determined by comparing RAP-PMR in the high-pressure zone (HPZ) and PAC. Contraction in the distal anal canal was interpreted as external anal sphincter contraction (EASC) and was compared with RAP-PMR in the HPZ. RESULTS: Relaxation was significantly greater in PAC than in HPZ (50 vs. 36 percent; P = 0.001). RAP-PMR was significantly higher in HPZ than in PAC (30.7 vs. 12.6 mmHg; P = 0.001). EASC was observed in six patients and did not show significant difference with RAP-PMR in HPZ (39.7 vs. 36.3 mmHg; not significant). Relaxation began at the same time in all levels but lasted significantly longer in PAC compared with HPZ (13.5 vs. 9.4 sec; P = 0.003). CONCLUSION: Anal relaxation induced by small volume rectal distention involves a gradient in the pressure and time of relaxation between PAC and the HPZ.

Adult↗

PubMed1987

The Rapid Acute Physiology Score.

The Rapid Acute Physiology Score (RAPS) was developed and tested for use as a severity scale in critical care transports. RAPS is an abbreviated version of the Acute Physiology and Chronic Health Evaluation (APACHE-II) using only parameters routinely available on all transported patients (i.e. pulse, blood pressure, respiratory rate, and Glasgow Coma Scale). RAPS has a range from 0 (normal) to 16. Two hundred eighty-three patients were transported by helicopter; 62 died. Pretransport RAPS was available on 282 of 283 patients (mean, 3.85; median, 3). Because of death, discharge, or transfer, 227 complete APACHE-II scores using least physiologic values for the first 24 hours after transfer were collected (mean, 14.98; median, 13). Stepwise logistic regression showed that when all APACHE-II and RAPS values were available, the best single predictor of mortality was worst value APACHE-II (X2(1) = 57.09, P less than .01). When pretransport RAPS was considered as a single explanatory variable, it too had significant predictive power for mortality (X2(1) = 92.53, P less than .01). Correlation analysis comparing RAPS with APACHE-II values at similar points in time revealed a significant relationship in all cases, with the highest correlation between RAPS worst values and APACHE-II worst values (r = .8472, P less than .01). It was concluded that RAPS can be applied usefully in complement with APACHE-II and may have limited utility when used alone.

Acute Disease↗

PubMed1997

Synergism between sirolimus and 1,25-dihydroxyvitamin D3 in vitro and in vivo.

The active form of vitamin D, 1 alpha, 25-(OH)2D3, displays immunomodulatory effects in vitro and in vivo at pharmacological levels. We evaluated the dose-effect relationship of 1,25(OH)2D3 and sirolimus (rapamycin, RAP) in vitro, on the inhibition of PHA-stimulated PBMC proliferation, by using the median effect analysis. Pharmacological concentrations of 1,25(OH)2D3 (between 10(-9) and 3 x 10(-6) M) interacted synergistically with RAP (combination index value of 0.01 for 50% suppression of PBMC proliferation). In vivo, the effect of 1,25(OH)2D3 and RAP combinations on the evolution of experimental allergic encephalomyelitis in SJL mice was analyzed. 1,25(OH)2D3, given i.p., in monotherapy, at a dose of 2 micrograms/kg every two days, from day -3 until day +19 after disease induction, or RAP, injected daily at a dose of 0.3 mg/kg for the same period, decreased EAE incidence (paralysis in 70 and 55% of the animals, respectively, versus 98% in the placebo treated group, p < 0.001). The combination treatment using the two drugs in these subtherapeutical doses provided near-total clinical (8% paralysis, p < 0.001 compared to monotherapy with 1,25(OH)2D3 or RAP) and histological protection, comparable to that obtained with RAP in monotherapy at a threefold higher dose (1 mg/kg/d). When the two drugs were given using an alternate day administration schedule (RAP at 0.6 mg/kg and 1,25(OH)2D3 at 2 micrograms/kg, each given on alternate days from day -3 to 19), near total protection was again obtained (13% paralysis, p < 0.001 versus control). These in vitro and in vivo data support the existence of synergistic interactions between 1,25(OH)2D3 and RAP. Considering the narrow therapeutic windows of both RAP and vitamin D-related compounds in autoimmune disease models, combinations of these drugs could find clinical application in reducing their individual therapeutically efficient doses to non-toxic levels.

Animals↗

PubMed1997

Receptor-associated protein in an oviparous species is correlated with the expression of a receptor variant.

The biosynthesis of proteins containing cysteine-rich domains requires chaperones for their correct folding. For instance, the 39-kDa receptor-associated protein (RAP) aides in the cell-surface targeting of newly synthesized members of the mammalian low density lipoprotein receptor (LDLR) gene family, which contains tandemly arranged clusters of hexacysteine repeats. In the chicken, an LDLR relative with eight such repeats is expressed as two different splice variant forms in cell type-specific fashion (Bujo, H., Lindstedt, K. A., Hermann, M., Mola Dalmau, L., Nimpf, J., and Schneider, W. J. (1995) J. Biol. Chem. 270, 23546-23551). To learn more about evolutionary aspects of RAP, its role in escorting of these different receptor splice variants, and other potential functions, we have extended our studies on the avian LDLR family to RAP. cDNA cloning, determination of tissue expression at both the transcript and the protein level, stable expression in COS cells, and binding studies with chicken RAP revealed that mammalian RAPs have retained many features of the non-amniotic proteins. However, structural details, e.g. the well defined internal triplicate repeats in the chicken protein, have been somewhat diluted during evolution. Interestingly, chicken RAP was found to correlate positively with the expression levels in somatic cells of the larger splice variant of the eight-cysteine repeat receptor, but not with those of the smaller variant, expressed only in germ cells. This is compatible with the possibility that RAP may play a role in receptor biology that could be complementing its function in assisting folding. Chicken RAP in crude extracts of the stable expressor COS cells is able to bind to LDLR relatives in ligand blots without requirement for prior purification of the ligand. Thus, in conjunction with the avian model of massive lipid transport to germ cells, these cells provide a novel comparative system amenable to investigation of the biological functions of RAP.

Amino Acid Sequence↗

PubMed2003

Allosteric modulation of ligand binding to low density lipoprotein receptor-related protein by the receptor-associated protein requires critical lysine residues within its carboxyl-terminal domain.

The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that recognizes more than 30 different ligands and plays important roles in protease and lipoprotein catabolism. Ligand binding to newly synthesized LRP is modulated by the receptor-associated protein (RAP), an endoplasmic reticulum-resident protein that functions as a molecular chaperone and prevents ligands from associating with LRP via an allosteric-type mechanism. RAP is a multidomain protein that contains two independent LRP binding sites, one located at the amino-terminal portion of the molecule and the other at the carboxyl-terminal portion of the molecule. The objective of the present investigation was to gain insight into how these two regions of RAP interact with LRP and function to modulate its ligand binding properties. These objectives were accomplished by random mutagenesis of RAP, which identified two critical lysine residues, Lys-256 and Lys-270, within the carboxyl-terminal domain that are necessary for binding of this region of RAP to LRP and to heparin. RAP molecules in which either of these two lysine residues was mutated still bound LRP but with reduced affinity. Furthermore, the mutant RAPs were significantly impaired in their ability to inhibit alpha(2)M* binding to LRP via allosteric mechanisms. In contrast, the mutant RAP molecules were still effective at inhibiting uPA.PAI-1 binding to LRP. These results confirm that both LRP binding sites within RAP cooperate to inhibit ligand binding via an allosteric mechanism.

Allosteric Site↗

PubMed2004

Rap1 regulates E-cadherin-mediated cell-cell adhesion.

The small GTPase Rap is best characterized as a critical regulator of integrin-mediated cell adhesion, although its mechanism of action is not understood. Rap also influences the properties of other cell-surface receptors and biological processes, although whether these are a consequence of effects on integrins is not clear. We show here that Rap also plays an important role in the regulation of cadherin-mediated cell-cell adhesion in epithelial cells. Expression of constitutively active Rap1A restored cadherin-mediated cell-cell contacts in mesenchymal Ras-transformed Madin-Darby canine kidney cells, resulting in reversion to an epithelial phenotype. Activation of endogenous Rap via the Rap exchange factor Epac1 also antagonized hepatocyte growth factor-induced disruption of adherens junctions. Inhibition of Rap signaling resulted in disruption of epithelial cell-cell contacts. Rap activity was required for adhesion of cells to recombinant E-cadherin extracellular domains, i.e. in the absence of integrin-mediated adhesion. These findings suggest that Rap signaling positively contributes to cadherin-mediated adhesion and that this occurs independently of effects on integrin-mediated adhesion. Our results imply that Rap may function in a broader manner to regulate the function of cell-surface adhesion receptors.

Animals↗

PubMed2001

Duration of action of vecuronium after an intubating dose of rapacuronium, vecuronium, or succinylcholine.

UNLABELLED: Rapacuronium (RAP) is a new, rapid-onset, short-duration, nondepolarizing neuromuscular blocker. If RAP is used to facilitate endotracheal intubation, what will the duration of a subsequent maintenance dose of vecuronium (VEC) be? We investigated the duration of action of a maintenance dose of VEC after intubation with RAP, VEC, or succinylcholine (SUC). Adult surgical patients under general anesthesia were randomly allocated to receive a tracheal intubating dose of RAP 1.5 mg/kg, VEC 0.1 mg/kg, or SUC 1 mg/kg. The anesthetic was induced with propofol and maintained with propofol, nitrous oxide, and oxygen. Neuromuscular function was monitored with electromyography. Recovery of the intubating dose of neuromuscular blocker was allowed to occur spontaneously until the first twitch of the train-of-four (T1) reached 50% of baseline, and then VEC 0.025 mg/kg (0.5 x 95% effective dose [ED(95)]) was administered. The onset, duration, and recovery to T1 = 25% and 50% were recorded. The durations of action (recovery of T1 25%) after intubating doses of RAP, VEC, and SUC were 13.7 +/- 5.3, 43.2 +/- 13.2, and 9.2 +/- 3.7 min (mean +/- SD), respectively (P < 0.0001). The times to maximum depression of T1 after a maintenance dose of VEC (0.5 x ED(95)) were 5.4 +/- 2.9, 5.1 +/- 2.5, and 5.3 +/- 2.8 min (mean +/- SD) for the RAP, VEC, and SUC groups, respectively. Recoveries to T1 25% after VEC for the RAP, VEC, and SUC groups were 18.9 +/- 11.5, 21.5 +/- 8.03, and 12.8 +/- 8.4 min, and at T1 50% they were 21.5 +/- 9.1, 30.8 +/- 9.5, and 15.5 +/- 9.7 min (mean +/- SD), respectively (P < 0.001, RAP and VEC versus SUC). The duration of action of a maintenance dose of VEC was similar after an intubating dose of RAP or VEC but was shortened when preceded by an intubating dose of SUC. IMPLICATIONS: The duration of action of a maintenance dose of vecuronium was longer after an endotracheal intubating dose of rapacuronium compared with succinylcholine.

Adolescent↗