Markers of hyperactive central corticotropin releasing factor (CRF) systems and CRF-related single nucleotide polymorphisms (SNPs) have been identified in patients with anxiety and depressive disorders. Designing more effective antagonists may now be guided by data showing that small molecules bind to transmembrane domains. Specifically, CRF1 receptor antagonists have been developed as novel anxiolytic and antidepressant treatments. Because CRF1 receptors become rapidly desensitized by G protein-coupled receptor kinase (GRK) and β-arrestin mechanisms in the presence of high agonist concentrations, neuronal hypersecretion of synaptic CRF alone may be insufficient to account for excessive central CRF neurotransmission in stress-induced affective pathophysiology. In addition to desensitizing receptor function, GRK phosphorylation and β-arrestin binding can shift a G protein-coupled receptor (GPCR) to signal selectively via the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) or Akt pathways independent of G proteins. Also, Epac-dependent CRF1 receptor signaling via the ERK-MAPK pathway has been found to potentiate brain-derived neurotrophic factor (BDNF)-stimulated TrkB signaling. Thus, genetic or acquired abnormalities in GRK and β-arrestin function may be involved in the pathophysiology of stress-induced anxiety and depression.

No abstract

Agonist-induced endocytosis and processing of the G protein–coupled AT1 angiotensin II (Ang II) receptor (AT1R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)– or hemagglutinin epitope–tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5–GFP and Rab4–GFP in early endosomes, and subsequently with Rab11–GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand–receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa®–transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine–Ang II was also segregated into small internal vesicles. In cells labeled with 125I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT1R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT1 receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase–dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.

Oakley RH, Olivares-Reyes JA, Hudson CC, Flores-Vega F, Dautzenberg FM, Hauger RL. Carboxyl terminal and intracellular loop sites for CRF1 receptor phosphorylation and ␤-arrestin-2 recruitment: a mechanism regulating stress and anxiety responses. Am J Physiol Regul Integr Comp Physiol 293: R209-R222, 2007. First published March 15, 2007; doi:10.1152/ajpregu.00099.2006.-The primary goal was to test the hypothesis that agonist-induced corticotropin-releasing factor type 1 (CRF1) receptor phosphorylation is required for ␤-arrestins to translocate from cytosol to the cell membrane. We also sought to determine the relative importance to ␤-arrestin recruitment of motifs in the CRF1 receptor carboxyl terminus and third intracellular loop. ␤-Arrestin-2 translocated significantly more rapidly than ␤-arrestin-1 to agonist-activated membrane CRF1 receptors in multiple cell lines. Although CRF1 receptors internalized with agonist treatment, neither arrestin isoform trafficked with the receptor inside the cell, indicating that CRF1 receptor-arrestin complexes dissociate at or near the cell membrane. Both arrestin and clathrin-dependent mechanisms were involved in CRF1 receptor internalization. To investigate molecular determinants mediating the robust ␤-arrestin-2-CRF1 receptor interaction, mutagenesis was performed to remove potential G protein-coupled receptor kinase phosphorylation sites. Truncating the CRF1 receptor carboxyl terminus at serine-386 greatly reduced agonist-dependent phosphorylation but only partially impaired ␤-arrestin-2 recruitment. Removal of a serine/ threonine cluster in the third intracellular loop also significantly reduced CRF1 receptor phosphorylation but did not alter ␤-arrestin-2 recruitment. Phosphorylation was abolished in a CRF1 receptor possessing both mutations. Surprisingly, this mutant still recruited ␤-arrestin-2. These mutations did not alter membrane expression or cAMP signaling of CRF 1 receptors. Our data reveal the involvement of at least the following two distinct receptor regions in ␤-arrestin-2 recruitment: 1) a carboxyl-terminal motif in which serine/threonine residues must be phosphorylated and 2) an intracellular loop motif configured by agonist-induced changes in CRF 1 receptor conformation. Deficient ␤-arrestin-2-CRF 1 receptor interactions could contribute to the pathophysiology of affective disorders by inducing excessive CRF 1 receptor signaling.corticotropin-releasing factor; G protein-coupled receptor kinase; receptor phosphorylation; internalization; stress adaptation THE MAGNITUDE AND DURATION of cellular signals transduced by G protein-coupled receptors (GPCRs) depend upon stringent regulation to prevent the deleterious effects of unrestrained receptor activation (12,26,27,31,45,50,55). Many studies have shown that agonist-induced GPCR signaling is rapidly attenuated by a mechanism termed "homologous desensitization." According to the classic model of homologous desensitization, agonist-activated receptors are phosphorylated by a family of G protein-coupled receptor...

Stimulation of the angiotensin II (Ang II) type 1 receptor (AT1-R) causes phosphorylation of extracellularly regulated kinases 1 and 2 (ERK1/2) via epidermal growth factor receptor (EGF-R) transactivation-dependent or -independent pathways in Ang II target cells. Here we examined the mechanisms involved in agonist-induced EGF-R transactivation and subsequent ERK1/2 phosphorylation in clone 9 (C9) hepatocytes, which express endogenous AT1-R, and COS-7 and human embryonic kidney (HEK) 293 cells transfected with the AT1-R. Ang II-induced ERK1/2 activation was attenuated by inhibition of Src kinase and of matrix metalloproteinases (MMPs) in C9 and COS-7 cells, but not in HEK 293 cells. Agonist-mediated MMP activation in C9 cells led to shedding of heparin-binding EGF (HB-EGF) and stimulation of ERK1/2 phosphorylation. Blockade of HB-EGF action by neutralizing antibody or its selective inhibitor, CRM197, attenuated ERK1/2 activation by Ang II. Consistent with its agonist action, HB-EGF stimulation of these cells caused marked phosphorylation of the EGF-R and its adapter molecule, Shc, as well as ERK1/2 and its dependent protein, p90 ribosomal S6 kinase, in a manner similar to that elicited by Ang II or EGF. Although the Tyr319 residue of the AT1-R has been proposed to be an essential regulator of EGF-R transactivation, stimulation of wild-type and mutant (Y319F) AT1-R expressed in COS-7 cells caused EGF-R transactivation and subsequent ERK1/2 phosphorylation through release of HB-EGF in a Src-dependent manner. In contrast, the noninvolvement of MMPs in HEK 293 cells, which may reflect the absence of Src activation by Ang II, was associated with lack of transactivation of the EGF-R. These data demonstrate that the individual actions of Ang II on EGF-R transactivation in specific cell types are related to differential involvement of MMP-dependent HB-EGF release.

The agonist-induced phosphorylation sites of the rat AT1a angiotensin receptor were analyzed using epitope-tagged mutant receptors expressed in Cos-7 cells. Angiotensin II-stimulated receptor phosphorylation was unaffected by truncation of the cytoplasmic tail of the receptor at Ser342 (Delta342) but was abolished by truncation at Ser325 (Delta325). Truncation at Ser335 (Delta335), or double-point mutations of Ser335 and Thr336 to alanine (ST-AA), reduced receptor phosphorylation by approximately 50%, indicating that in addition to Ser335 and/or Thr336, amino acids within the Ser326-Thr332 segment are also phosphorylated. Agonist-induced phosphorylation of the ST-AA and Delta335 receptors was partially inhibited by staurosporine, suggesting that the single protein kinase C consensus site in the Ser326-Thr332 segment (Ser331) is phosphorylated. The impairment of receptor phosphorylation was broadly correlated with the attenuation of agonist-induced internalization rates (Delta325 < Delta335 < ST-AA < Delta342 < wild-type) and with the increasing rank order of magnitude of inositol phosphate production normalized to an equal number of receptors (Delta325 > Delta335 > ST-AA = Delta342 > wild-type). These results demonstrate that agonist-induced phosphorylation of the AT1a receptor is confined to an 11-amino-acid serine/threonine-rich segment of its carboxyl-terminal cytoplasmic tail and implicate this region in the mechanisms of receptor internalization and desensitization.

In rat hepatic C9 cells, angiotensin II (Ang II)-induced activation of angiotensin type 1 (AT 1 ) receptors (AT 1 -Rs) stimulates extracellular signal-regulated kinase (ERK) 1/2 phosphorylation via transactivation of the endogenous epidermal growth factor (EGF) receptor (EGF-R) by a protein kinase C (PKC) ␦/Src/Pyk2-dependent pathway. This leads to phosphorylation of the EGF-R as well as its subsequent internalization. On the other hand, EGF-induced activation of the EGF-R in C9 cells was found to cause phosphorylation of the AT 1 -R. This was prevented by selective inhibition of the intrinsic tyrosine kinase activity of the EGF-R by AG1478 [4-(3Ј-chloroanilino)-6,7-dimethoxy-quinazoline] and was reduced by inhibition of PKC and phosphoinositide 3-kinase. EGF-induced AT 1 -R phosphorylation was associated with a decrease in membrane-associated AT 1 -Rs and a reduced inositol phosphate response to Ang II. Agonist activation of endogenous AT 1 -Rs and EGF-Rs induced the formation of a multireceptor complex containing both the AT 1 -R and the transactivated EGF-R. The dependence of these responses on caveolin was indicated by the finding that cholesterol depletion of C9 cells abolished Ang II-induced inositol phosphate production, activation of Akt/PKB and ERK1/2, and AT 1 -R internalization. Confocal microscopy demonstrated that caveolin-1 was endogenously phosphorylated and was distributed on the plasma membrane in patches that undergo redistribution during Ang II stimulation. Agonist-induced phosphorylation and association of caveolin 1 with the AT 1 -R was observed, consistent with a scaffolding role of caveolin during transactivation of the EGF-R by Ang II. The EGF-induced AT 1 -R/caveolin association was abolished by AG1478, suggesting that activation of the EGF-R promotes the association of caveolin and the AT 1 -R.

No abstract