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Functional activity and regulation of human beta 2-adrenergic receptors expressed in Xenopus oocytes
B K Kobilka1, C MacGregor, K Daniel
1Department of Medicine, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
Abstract:
The recently cloned human beta-adrenergic cDNA and several mutated forms have been expressed in Xenopus laevis oocytes by injection of RNA made from the cDNA under the control of the bacteriophage SP6 promoter. The cDNA and gene of the beta 2-adrenergic receptor possess the unusual feature of having a second upstream ATG (-101 base pairs) and a 19-codon open reading frame 5' to the initiator methionine codon of the receptor (Kobilka, B. K., Dixon, R. A. F., Frielle, T., Dohlman, H. G., Bolanowski, M., Sigal, I. S., Yang-Feng, T. L., Francke, U., Caron, M. G., and Lefkowitz, R. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 46-50). RNA lacking this upstream AUG and open reading frame was translated approximately 10-fold more efficiently both in an in vitro rabbit reticulocyte system and in oocytes. Injected oocytes but not water injected controls expressed typical beta 2-adrenergic receptors as assessed by ligand binding (450 fmol/mg membrane protein) and catecholamine-stimulated adenylate cyclase (approximately 20 fold). Moreover, these receptors displayed typical agonist-induced homologous desensitization when oocytes were incubated with isoproterenol at room temperature for 3-24 h. Among a series of mutations, truncations of the membrane-anchored core of the receptor eliminated receptor binding and cyclase stimulating activity. In contrast, disruption of one of the cAMP-dependent protein kinase phosphorylation sites or removal of the serine/threonine-rich carboxyl terminus had little or no effect on these functions or on the extent of agonist-induced desensitization relative to that observed with native receptor. These studies validate the beta 2-adrenergic nature of the cloned human beta-adrenergic cDNA, document the utility of the Xenopus oocyte system for studying functional and regulatory properties of receptors coupled to adenylate cyclase, and suggest the possibility that elements in the 5' untranslated region of the beta 2-adrenergic receptor RNA may regulate its translation in vivo.
Insights
Researchers expressed human beta-2 adrenergic receptors in frog oocytes, finding that removing a 5' untranslated region segment enhanced receptor translation and function. This study validates the receptor
Area of Science:
- Molecular Biology
- Pharmacology
- Cell Biology
Background:
- The human beta-2 adrenergic receptor (β2AR) is a key G protein-coupled receptor involved in various physiological processes.
- Understanding the regulation of β2AR expression and function is crucial for developing targeted therapies.
- Previous studies identified an unusual upstream open reading frame (uORF) in the β2AR mRNA, but its functional significance remained unclear.
Purpose of the Study:
- To investigate the functional role of the upstream open reading frame (uORF) in the 5' untranslated region (5'-UTR) of the human beta-2 adrenergic receptor (β2AR) mRNA.
- To validate the cloned human β2AR cDNA and assess the utility of the Xenopus oocyte expression system for studying receptor function and regulation.
- To explore the impact of specific mutations on β2AR activity, ligand binding, and desensitization.
Main Methods:
- RNA synthesis from cloned human β2AR cDNA and mutated forms using the SP6 promoter.
- Injection of synthesized RNA into Xenopus laevis oocytes for protein expression.
- Ligand binding assays to quantify receptor expression.
- Measurement of catecholamine-stimulated adenylate cyclase activity.
- Assessment of agonist-induced homologous desensitization following isoproterenol treatment.
Main Results:
- Expression of typical β2ARs in oocytes, confirmed by ligand binding and functional adenylate cyclase assays.
- RNA lacking the upstream AUG and 19-codon uORF showed approximately 10-fold increased translation efficiency in vitro and in oocytes.
- Receptors exhibited typical agonist-induced homologous desensitization.
- Truncations of the receptor core abolished binding and cyclase activity, while mutations in phosphorylation sites or the carboxyl terminus had minimal effects on function and desensitization.
Conclusions:
- The cloned human β2AR cDNA encodes a functional receptor that can be expressed and studied in Xenopus oocytes.
- The upstream open reading frame (uORF) in the 5'-UTR significantly regulates the translation of β2AR mRNA.
- Specific domains of the β2AR are critical for its function, while others, like phosphorylation sites, play less critical roles in agonist-induced desensitization.