Chimeric exchanges within the bradykinin B2 receptor intracellular face with the prostaglandin EP2 receptor as the donor: importance of the second intracellular loop for cAMP synthesis
| Autor: | Gregory N. Prado, Ruma Pal-Ghosh, Jun Yu, Linda Taylor, Peter Polgar, Dale F. Mierke |
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| Rok vydání: | 2003 |
| Předmět: |
Models
Molecular Gs alpha subunit Receptor Bradykinin B2 Macromolecular Substances Protein Conformation G protein Prostaglandin E2 receptor Molecular Sequence Data Gi alpha subunit Biophysics Biology Transfection Biochemistry Cyclic AMP Animals Humans Receptors Prostaglandin E Amino Acid Sequence Bradykinin receptor Receptor Molecular Biology Cells Cultured G protein-coupled receptor Receptors Bradykinin Stereoisomerism Intracellular Membranes Fibroblasts Receptors Prostaglandin E EP2 Subtype Recombinant Proteins Protein Structure Tertiary Rats Cell biology Mutagenesis Site-Directed Prostaglandin EP2 receptor Protein Binding |
| Zdroj: | Archives of Biochemistry and Biophysics. 415:54-62 |
| ISSN: | 0003-9861 |
| Popis: | The prostaglandin E2 (PGE(2)) EP2 receptor (EP2R) type is G protein coupled (GPCR) and links to Galphas. Through this receptor PGE(2) activates cAMP production. The bradykinin (BK) B2 receptor (BKB2R) is also a GPCR but links to Galphaq and Galphai and does not activate cAMP production in response to bradykinin. In an attempt to convert the BKB2R into a Galphas-linked adenylate cyclase-activating receptor we proceeded to make global and discrete motif replacements of the intracellular (IC) face of the BKB2R with the corresponding regions of the human EP2R. With this approach we produced hybrid receptors which, when stably transfected into wild type (WT) Rat-1 cells, bound BK but produced cAMP. Replacement of the second loop (IC2), third loop (IC3), the entire C terminus, and the distal C terminus resulted in receptors which bound BK. However, only the IC2 and IC3 exchanges resulted in cAMP-producing receptors. Of these two regions, the IC2 exchange was by far the better cAMP-generating receptor, producing cAMP at approximately 6.6-fold above WT BKB2R or approximately one fourth the amount produced by WT EP2R-transfected Rat-1 cells. Both human and rat EP2R and human beta2-adrenergic receptor exchanges of the IC2 produced equal quantities of cAMP. Focusing on the rBKB2R/hEP2R IC2 chimeras, the region consisting of residues 136-147 (BKB2R residue numbering) proved to contain a cAMP-generating motif. Within this region, the proximal six amino acids from the EP2R (HPYFYQ) at position 136-141 proved crucial for cAMP production (10-fold over WT BKB2R). The distal part of this region, the six residues at 142-147, played no role in cAMP production. On the other hand, the ALV motif of the BKB2R IC2, residues 133-135, proved important with respect to phosphatydilinositol (PI) turnover. Replacing the entire IC2 of BKB2R resulted in poor PI turnover, while including the AVL of BKB2R retained approximately half of the WT PI turnover. With respect to receptor uptake, all the IC2 mutants endocytosed as WT BKB2R (60% in 1h). However, the exchange of the distal and the whole C termini resulted in a marked drop in endocytosis (30% in 1h). These results demonstrate that the construction of a cAMP-producing BKB2/EP2 receptor hybrid is possible, with the IC2 region distal to DRYLALV proving important to Galphas linkage and the LALV motif within the IC2 of BKB2R and the region proximal to it proving important for Galphaq and Galphai linkage. Additionally, our results confirm the importance of the distal C terminus in determining receptor uptake. |
| Databáze: | OpenAIRE |
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