The preglomerular microcirculation of spontaneously hypertensive rats (SHR) is hypersensitive to angiotensin II, and studies show that this is likely due to enhanced convergent signaling between G-protein subunits αq (Gαq; released by angiotensin II) and G-protein subunits βγ (Gβγ; released by Gi-coupled receptors) to active phospholipase C (PLC). Here we investigated the molecular basis for the enhanced convergent signaling between Gβγ and Gαq in SHR preglomerular vascular smooth muscle cells (PGVSMCs). Because receptor for activated C kinase 1 (RACK1; scaffolding protein) organizes interactions between Gβγ, Gαq and PLC, we included RACK1 in this investigation. Cell fractionation studies demonstrated increased levels of membrane (but not cytosolic) Gβ, Gαq, PLCβ3 and RACK1 in SHR PGVSMCs compared with Wistar-Kyoto rat PGVSMCs. In SHR PGVSMCs, co-immunoprecipitation demonstrated RACK1 binding to Gβ and PLCβ3, but only at cell membranes. Pertussis toxin (blocks Gβγ) and U73122 (blocks PLC) reduced membrane RACK1; however, RACK1 knockdown (shRNA) did not affect membrane levels of Gβ, Gαq or PLCβ3. In a novel gel contraction assay RACK1 knockdown in SHR PGVSMCs attenuated contractions to angiotensin II and abrogated the ability of neuropeptide Y (signals via Gβγ) to enhance angiotensin II-induced contractions. We conclude that in SHR PGVSMCs the enlarged pool of Gβγ and PLCβ3 recruits RACK1 to membranes and RACK1 then organizes signaling. Consequently, knockdown of RACK1 prevents convergent signaling between angiotensin II and the Gi-pathway. This is the first study to implicate RACK1 in vascular smooth muscle cell contraction and suggests that RACK1 inhibitors could be effective cardiovascular drugs.
- scaffolding protein
- Copyright © 2016, American Journal of Physiology-Renal Physiology