Isoform-Dependent Loss- and Gain-of-Function of the Gαs K53N Variant in Human Disease

Abstract

The K53N mutation in Gs has been identified in patients with Albrights Hereditary Osteodystrophy (AHO), pseudohypoparathyroidism type 1A (PHP1a), and dilated cardiomyopathy; however, its molecular mechanism remains unclear. Here, we characterize the molecular, cellular, and physiological consequences of the K53N mutation in both long and short isoform of Gs. Biochemical analyses reveal that K53N disrupts nucleotide exchange and GTP hydrolysis, rendering both the short (Gs-S) and long (Gs-L) isoforms unresponsive to activation by G protein-coupled receptors (GPCRs) or cholera toxin. Both isoforms display a loss-of-function phenotype, failing to trigger cAMP production in response to {beta}2-adrenergic, parathyroid hormone, or vasopressin receptor stimulation. Notably, only the long isoform (Gs-L K53N) displays constitutive, receptor-independent cAMP generation. The mutation also reduces protein stability, weakens G{beta}{gamma} subunit interaction, and reduces plasma membrane localization. In neonatal rat ventricular cardiomyocytes, K53N impairs cAMP signaling and exerts dominant-negative effects on isoproterenol-induced responses. Strikingly, only Gs-L K53N abolishes isoproterenol-stimulated calcium release, directly implicating this isoform in the pathogenesis of cardiomyopathy. Collectively, these findings identify K53N as a unique Gs mutation that confers both loss- and gain-of-function properties in an isoform-specific manner, providing mechanistic insight into its complex pathogenicity in endocrine and cardiac tissues.