Molecular dissection of G protein preference using Gsalpha chimeras reveals novel ligand signaling of GPCRs.
Although only 16 genes have been identified in mammals, several Galpha subunits can be simultaneously activated by G protein-coupled receptors (GPCRs) to modulate their complicated functions. Current GPCR assays are limited in the evaluation of selective Galpha activation, thus not allowing a comprehensive pathway screening. Because adenylyl cyclases are directly activated by G(s)alpha and the carboxyl termini of the various Galpha proteins determine their receptor coupling specificity, we proposed a set of chimeric G(s)alpha where the COOH-terminal five amino acids are replaced by those of other Galpha proteins and used these to dissect the potential Galpha linked to a given GPCR. Unlike G(q)alpha, G(12)alpha, and G(i)alpha outputs, compounding the signals from several Galpha members, the chimeric G(s)alpha proteins provide a superior molecular approach that reflects the previously uncharacterized pathways of GPCRs under the same cAMP platform. This is, to our knowledge, the first time allowing verification of the whole spectrum of Galpha coupling preference of adenosine A1 receptor, reported to couple to multiple G proteins and modulate many physiological processes. Furthermore, we were able to distinguish the uncharacterized pathways between the two neuromedin U receptors (NMURs), which distribute differently but are stimulated by a common agonist. In contrast to the G(q) signals mainly conducted by NMUR1, NMUR2 routed preferentially to the G(i) pathways. Dissecting the potential Galpha coupling to these GPCRs will promote an understanding of their physiological roles and benefit the pharmaceutical development of agonists/antagonists by exploiting the selective affinity toward a certain Galpha subclass.
Hsu
Shih-Han
SH
Dept. of Life Sciences and Inst. of Genome Sciences, National Yang-Ming University, 155 Li Nong St., Section 2, Shihpai, Taipei 112, Taiwan.
Luo
Ching-Wei
CW
D000818:Animals; D002478:Cells, Cultured; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D040081:Protein Array Analysis; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D043682:Receptor, Adenosine A1 / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013379:Substrate Specificity; D014162:Transfection
D044385:GTP-Binding Protein alpha Subunits; D008024:Ligands; D043682:Receptor, Adenosine A1; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins