A novel assay to diagnose hereditary angioedema utilizing inhibition of bradykinin-forming enzymes.

BACKGROUND: Hereditary angioedema types I and II are caused by a functional deficiency of C1 inhibitor (C1-INH), leading to overproduction of bradykinin. The current functional diagnostic assays employ inhibition of activated C1s; however, an alternative, more physiologic method is desirable. METHODS: ELISAs were developed using biotinylated activated factor XII (factor XIIa) or biotinylated kallikrein bound to avidin-coated plates. Incubation with plasma was followed by detection of bound C1-INH. RESULTS: After standard curves were developed for quantification of C1-INH, serial dilutions of normal plasma were employed to validate the ability to detect known concentration of C1-INH in the plasma as a percent of normal. Hereditary angioedema (HAE) types I and II were then tested. The level of functional C1-INH in all HAE types I and II plasma tested was less than 40% of our normal control. This was evident regardless of whether we measured factor XIIa-C1-INH or kallikrein-C1-INH complexes, and the two assays were in close agreement. By contrast, testing the same samples utilizing the commercial method (complex ELISA, Quidel Corp.) revealed the levels of C1-INH between 0 and 57% of normal (mean, 38%), and 42 samples were considered equivocal (four controls and 38 patients). CONCLUSIONS: Diagnosis of HAE types I and II can be ascertained by inhibition of enzymes of the bradykinin-forming cascade, namely factor XIIa and kallikrein. Either method yields functional C1-INH levels in patients with HAE (types I and II) that are clearly abnormal with less variance or uncertainty than the commercial method.

Bradykinin B2 receptors activate Na+/H+ exchange in mIMCD-3 cells via Janus kinase 2 and Ca2+/calmodulin.

We used a cultured murine cell model of the inner medullary collecting duct (mIMCD-3 cells) to examine the regulation of the ubiquitous sodium-proton exchanger, Na+/H+ exchanger isoform 1 (NHE-1), by a prototypical G protein-coupled receptor, the bradykinin B2 receptor. Bradykinin rapidly activates NHE-1 in a concentration-dependent manner as assessed by proton microphysiometry of quiescent cells and by 2'-7'-bis[2-carboxymethyl]-5(6)-carboxyfluorescein fluorescence measuring the accelerated rate of pH(i) recovery from an imposed acid load. The activation of NHE-1 is blocked by inhibitors of the bradykinin B2 receptor, phospholipase C, Ca2+/calmodulin (CaM), and Janus kinase 2 (Jak2), but not by pertussis toxin or by inhibitors of protein kinase C and phosphatidylinositol 3'-kinase. Immunoprecipitation studies showed that bradykinin stimulates the assembly of a signal transduction complex that includes CaM, Jak2, and NHE-1. CaM appears to be a direct substrate for phosphorylation by Jak2 as measured by an in vitro kinase assay. We propose that Jak2 is a new indirect regulator of NHE-1 activity, which modulates the activity of NHE-1 by increasing the tyrosine phosphorylation of CaM and most likely by increasing the binding of CaM to NHE-1.

Analysis of the AU-rich elements in the 3'-untranslated region of beta 2-adrenergic receptor mRNA by mutagenesis and identification of the homologous AU-rich region from different species.

The 35000-Mr beta-adrenergic receptor mRNA binding protein (beta ARB) is induced by beta-adrenergic agonists and binds to G-protein-linked receptor mRNAs that exhibit agonist-induced destabilization. Recently, we identified a 20-nucleotide, AU-rich region in the 3'-untranslated region of the hamster beta 2-adrenergic receptor mRNA consisting of an AUUUUA hexamer flanked by U-rich regions, which constitutes the binding domain for beta ARB. U to G substitution in the hexamer region attenuates the binding of beta ARB, whereas U to G substitution of hexamer and flanking U-rich domains abolishes binding of beta ARB and stabilizes beta 2-adrenergic receptor mRNA levels in transfectant clones challenged with either isoproterenol or cyclic AMP. In the study presented here, we mutated the 20-nucleotide ARE region to establish the minimal AU-rich sequence required for beta ARB binding. U to G substitutions of flanking poly(U) regions and of the hexamer established the nature of the binding properties. Using various mutants, we demonstrated also that binding of beta ARB correlates with the extent of destabilization of beta 2-adrenergic receptor mRNA in response to agonist stimulation. High-affinity binding of hamster, rat, mouse, porcine, and human ARE sequences to beta ARB was revealed by SDS-polyacrylamide gel electrophoresis following UV-catalyzed cross-linking and by gel mobility shift assays. Further, beta ARB was shown to bind more avidly to the 20-nucleotide ARE region than to well-established mRNA destablization sequences of tandem repeats of five pentamers. Thus, for beta 2-adrenergic receptor, mRNA destabilization likely occurs via conserved AU-rich elements present in the 3'-untranslated regions of receptor mRNAs.

A 20-nucleotide (A + U)-rich element of beta2-adrenergic receptor (beta2AR) mRNA mediates binding to beta2AR-binding protein and is obligate for agonist-induced destabilization of receptor mRNA.

The Mr 35,000 beta-adrenergic receptor mRNA-binding protein, termed betaARB protein, is induced by beta-adrenergic agonists and binds to beta2-receptor mRNAs that display agonist-induced destabilization. A cognate sequence in the mRNA was identified previously that provides for betaARB protein binding in vitro. In the present work, the sequence established in vitro for binding of betaARB protein to hamster beta2-adrenergic receptor mRNA was probed in vivo by site-directed mutagenesis of the 3'-untranslated region and expression in Chinese hamster ovary cells. A 20-nucleotide, (A + U)-rich region in the 3'-untranslated region consisting of an AUUUUA hexamer flanked by defined U-rich regions constitutes the binding domain for betaARB protein. U to G substitution in the hexamer region attenuates the binding of betaARB protein, whereas U to G substitution of hexamer and flanking U-rich domains abolishes binding of betaARB protein and stabilizes beta2-adrenergic receptor mRNA levels in transfectant clones challenged with either isoproterenol or cyclic AMP. These results demonstrate that binding of betaARB protein to the 20-nucleotide, (A + U)-rich domain mediates the agonist and cyclic AMP-induced mRNA decay of G protein-linked receptors, such as the beta2-adrenergic receptor.

The M(r) 35,000 beta-adrenergic receptor mRNA-binding protein binds transcripts of G-protein-linked receptors which undergo agonist-induced destabilization.

The M(r) 35,000 beta-adrenergic receptor mRNA-binding protein, termed beta-ARB protein, is induced by beta-adrenergic agonists and binds to beta 2-receptor mRNAs that display agonist-induced destabilization. Recently a cognate sequence in the mRNA was identified that provides for recognition by beta-ARB protein. In the present work we test the ability of the beta-ARB to discriminate among G-protein-linked receptor mRNAs that either do or do not display agonist-induced destabilization and test the predictive value of the presence of the cognate sequence to identify receptors displaying post-transcriptional regulation. Transcripts of beta 2-, but not rat beta 1-, rat beta 3-, or human beta 3- adrenergic receptors bind beta-ARB protein, linking agonist-induced destabilization of mRNA to transcripts with the cognate sequence. Scanning GeneBank for G-protein-linked receptor transcripts with the cognate sequence revealed several candidates, including the thrombin receptor. We demonstrate that the thrombin receptor mRNA is recognized by beta-ARB protein and like the beta 2-receptor is regulated post-transcriptionally by agonist and cAMP. Thus, the domain of regulation by beta-ARB protein includes transcripts of G-protein-linked receptors other than beta 2-adrenergic receptors.

The M(r) 35,000 beta-adrenergic receptor mRNA-binding protein induced by agonists requires both an AUUUA pentamer and U-rich domains for RNA recognition.

Delineating the molecular basis for agonist-induced destabilization of mRNA of G-protein-linked receptors that contributes to receptor down-regulation is fundamental to our understanding of long-term regulation of receptors by agonist. Previously we identified a prominent, M(r) 35,000 cytosolic RNA-binding protein that (i) binds selectively to beta 1 and beta 2-adrenergic receptor mRNAs, both of which undergo agonist-induced down-regulation; (ii) does not bind either to alpha 1b-adrenergic receptor mRNA, which does not undergo agonist-induced down-regulation, or to beta-globin mRNA; (iii) displays binding to beta 2-adrenergic receptor mRNA that is selectively competed by poly(U) RNA, but not poly(A),-(C), or -(G) RNA; and (iv) its abundance varies inversely with the level of receptor mRNA, being induced by agonists that down-regulate receptor mRNA (Port, J. D., Huang, L.-y., and Malbon (1992) J. Biol. Chem. 267, 24103-24108). We demonstrate here that the binding of beta-adrenergic receptor mRNA by this protein, termed beta-ARB protein, is sensitive to competition by AU-rich domains of the 3'-untranslated regions of c-fos, c-myc, and human granulocyte-macrophage colony-stimulating factor. Using the AU-rich 3'-untranslated regions of wild-type adenovirus IVa2 mRNA and variants with defined mutations in the AUUUApentamer, AU-rich, and U-rich domains, we were able to define sequences critical to the binding of the beta 2-receptor mRNA by the beta-ARB protein. Recognition of beta-ARB protein requires not only an AUUUA destabilization pentamer, but also a flanking U-rich domain(s). Using radiolabeled 3'-untranslated regions of short-lived mRNA, we were able to identify this same M(r) 35,000 cytosolic RNA-binding protein(s), beta-ARB protein, as selective for beta 2-adrenergic receptor mRNA.