ANALYSIS OF AGONIST FUNCTION AT FUSION PROTEINS BETWEEN THE IP PROSTANOID RECEPTOR AND COGNATE, UNNATURAL AND CHIMAERIC G-PROTEINS

摘要: Direct measures of G-protein activation based on guanine nucleotide exchange and hydrolysis are frequently impossible to monitor for receptors which interact predominantly with G(s)alpha. An isolated FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys)-epitope-tagged human IP prostanoid receptor fusion proteins generated between this form the alpha subunits its cognate G(s), G(i1), a it fails activate in co-expression studies, chimaeric G(i1)-G(s)6 (a G(i1) C-terminal six amino acids were replaced equivalent sequence G(s)) stably expressed HEK293 cells. These detected by [(3)H]ligand-binding studies immunoblotting both an anti-FLAG antibody appropriate antisera G-proteins. Each construct displayed similar affinity bind agonist iloprost. Iloprost stimulated adenylate cyclase activity clones expressing receptor-G(s)alpha protein, constructs shown endogenously Addition iloprost membranes cells resulted small stimulation high-affinity GTPase activity. produced no could be attributed receptor-G(i1)alpha fusion. However, containing either G(s)alpha or G(i1)-G(s)6alpha substantially greater than receptor. Treatment receptor-G(i1)-G(s)6alpha protein combination cholera pertussis toxins allowed direct measurement receptor-linked G-protein. Normalization such results levels expression demonstrated 5-fold higher when using G(s)alpha-containing 9-fold improvement detect compared