Rapid desensitization of neonatal rat liver beta-adrenergic receptors. A role for beta-adrenergic receptor kinase.

Abstract

Exposure of fl-adrenergic receptors (BAR) to agonists often leads to a rapid loss of receptor responsiveness. The proposed mechanisms of such rapid receptor desensitization include receptor phosphorylation by either cAMP-dependent protein kinase or the specific 8-adrenergic receptor kinase (BARK), leading to functional uncoupling from adenylyl cyclase and sequestration of the receptors away from the cell surface. To evaluate the physiological role of such mechanisms, we have investigated whether rapid regulation of BAR occurs in the neonatal rat liver immediately after birth, a physiological situation characterized by a dramatic but transient increase in plasma catecholamines. We have detected a rapid, transient uncoupling of liver plasma membrane BARs from adenylyl cyclase (corresponding to a desensitization of -45%) within the first minutes of extrauterine life, followed by a transient sequestration of -40% of the BARs away from the plasma membrane. In agreement with such pattern of desensitization, we have detected (by enzymatic and immunological assays) rapid changes in BARK specific activity in different neonatal rat liver subcellular fractions that take place within the same time frame of BAR uncoupling and sequestration. Our results provide new evidence on the potential role of BAR desensitization mechanisms in vivo and suggest that they are involved in modulating catecholamines actions at the moment of birth. Furthermore, our data indicate that in addition to its suggested role as a rapid modulator of adrenergic receptor function at synapse, rapid BARK-mediated receptor regulation may have functional relevance in other tissues in response to high circulating or local levels of agonists. (J. Clin. Invest. 1994. 93:937-943.)

G Alpha-q/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

“…To determine whether G␤␥ subunits were involved in glucose transport signaling, we used a glutathione S-transferase (GST) fusion protein containing the C-terminal portion of the ␤-adrenergic receptor kinase (GST-BARK). This protein binds to G␤␥ subunits and behaves as a dominant negative inhibitor of G␤␥ signaling (7). We therefore microinjected GST-BARK into the cytoplasm of cells infected with WT or Q209L-G␣q adenovirus and measured insulin-stimulated GLUT4 translocation.…”

Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2

“…Gels were loaded with 5 g of cell lysate proteins from transiently transfected HEK293 cells or with 40 g of protein from Mono Mac 1 cell lysates or Mono Mac 1 subcellular fractions. In some experiments, soluble and particulate Mono Mac 1 cell fractions were obtained under different experimental conditions, and the subcellular GRK2 distribution was assessed in Western blots as reported (32). After fractionation the protein concentration in each sample was determined by the Lowry method and equal amounts of protein were loaded onto the gels.…”

G Protein–coupled Receptor Kinases

“…These investigations can be categorized broadly as studies that (a) evaluate the contribution of GRK action, among other mechanisms, to the desensitization of a particular receptor; (b) investigate the kinetics of GRK-initiated desensitization; (c) define GRK subcellular localization; (d ) assay factors affecting GRK expression or activity; and (e) evaluate candidate receptor substrates. Although the preponderance of these studies employ immortalized cell lines, several of the conclusions derived from these cell lines have been corroborated by studies performed in primary cultures of cells (105,(154)(155)(156)(157)(158), the constituents of which more closely model in vivo conditions. In immortalized cell lines (159)(160)(161)(162) as well as in primaryculture models (9,105,129,163,164), there may be considerable cell typespecific variation in relative and even absolute GRK expression.…”