A Gαsand GαiChimera Alters cAMP Kinetics and Appears to Inhibit GRK Function

Abstract ID 98451 Poster Board 158 Canonically, GPCRs interact with the GDP-bound heterotrimer G protein (Gαβγ) in a cytosolic pocket in the receptor, and when signalling is terminated the Gαβγ return to the receptor. GRKs utilize the same pocket to interact with the active GPCR. We hypothesize that the returning inactive Gαβγ and GRKs compete for the same binding site if the receptor remains in the active conformation. To investigate this, a chimera containing a portion of Gαi1 was inserted into the first half of the helical domain of Gαs (Gs-60-Gi1-138-Gs), named sis. To exclude background effects of endogenous Gαs, GnasE2-/E2- MEFs with endogenous β2-adrenergic receptors (β2-ARs) were transfected with Gαs or sis and a split luciferase cAMP probe. The difference between 1 μM isoproterenol (ISO)-mediated Gαs and sis cAMP accumulation and decay kinetics was investigated. The accumulation kinetics demonstrate that sis produces more cAMP (Gαs = 2.4-fold & sis = 3.1-fold, p = 0.005) but at a slower rate (Tau: Gαs = 3.4 & sis = 4.8 min, p = 0.003), and the decay kinetics were drastically different. The cAMP decay kinetics of sis-expressing cells fits zero order kinetics versus first order kinetics for Gαs cells (p = 0.0014), resulting in a much slower decay (Tau: Gαs = 22.8 ± 12.6 & sis = 57.9 ± 24.7 min). To determine if the change in decay kinetics is due to prolonged receptor activation, 10 μM propranolol (PROP) was added 12 minutes after ISO stimulation. PROP induced sis cAMP decay kinetics to follow first-order kinetics (p = 0.0070), resulting in sis reaching baseline (Tau: 29.1 ± 13.3 min, p = 0.0117 vs sis w/o PROP). The PROP-induced changes indicate that the sis construct allows the β2-ARs to remain in an agonist-bound active state for a longer duration. Transfection of GRK2,4,5, and 6 failed to decrease ISO-mediated cAMP production in sis cells; therefore, 10 μM GRK inhibitor (CMPD101) was used to observe the role of GRKs in Gαs-mediated cAMP production. Although underpowered, the data demonstrated that inhibition of GRKs changed half the Gαs samples cAMP decay kinetics to zero-order linear kinetics (p = 0.2821); however, more experiments are being conducted to match a power analysis. To determine if sis cycles rapidly, NanoLuc was inserted into Gαs and sis to examine Gαβγ association and dissociation with a split Venus Gβ and Gγ via BRET; these experiments are ongoing. The data supports the hypothesis that increasing the kinetics of G protein cycling interfere with GRK-mediated inhibition of canonical receptor signalling. WesternU MSBPS program funds and NIH grant DK121776 (MB) supported this work.