G-protein-coupled receptor kinase 3- and protein kinase C-mediated desensitization of the PACAP receptor type 1 in human Y-79 retinoblastoma cells
Introduction
Pituitary adenylyl cyclase-activating polypeptide (PACAP), a neuropeptide released by the hypothalamus, was originally identified by its property of dramatically stimulating adenylyl cyclase activity in anterior pituitary cells (Arimura and Shioda, 1995, Vaudry et al., 2000). PACAP belongs to the peptide family that includes vasoactive intestinal polypeptide (VIP), secretin, glucagon, growth hormone releasing factor (GRF) and corticotropin-releasing factor (CRF). In addition to functioning as a hypophysiotropic hormone, neurotransmitter and neuromodulator (reviewed in Arimura and Shioda, 1995, and Vaudry et al., 2000), PACAP plays a critical role in neurogenesis (Deutsch and Sun, 1992, Waschek et al., 1998). Endogenous, post-translational modification of the PACAP precursor protein produces two forms of the peptide: PACAP27 and PACAP38. PACAP27, a C-terminally shortened version of the longer PACAP38 (Arimura and Shioda, 1995), shares 68% sequence homology with VIP. Both forms of the PACAP peptide bind to three related, but structurally unique, PACAP receptors: PAC1, VPAC1, and VPAC2. These three receptors belong to the class B subfamily of G-protein-coupled receptors (GPCRs) that signal via Gs, the stimulatory heterotrimeric GTP-binding protein. In addition to the three PACAP receptors, this subfamily includes the secretin, glucagon, calcitonin, GRF and CRF receptors. PAC1, VPAC1, and VPAC2 differ from one another in their ligand binding and selectivity properties, and in their tissue distribution.
Although the PAC1 receptor is expressed abundantly in the central nervous system, its presence in peripheral organs is limited (reviewed in Harmar and Lutz, 1994, and Vaudry et al., 2000). Both the VPAC1 and VPAC2 receptors, on the other hand, are widely distributed in both the brain and periphery (Harmar and Lutz, 1994, Vaudry et al., 2000). All three receptor subtypes have been found in retina (D'Agata and Cavallaro, 1998). VPAC1 and VPAC2 nonselectively bind PACAP38, PACAP27 and VIP with high affinity (Harmar and Lutz, 1994). In contrast, the ligand selectivity profile of PAC1, which binds PACAP38 and PACAP27 with more than 100-fold greater affinity than VIP, is significantly narrower (Spengler et al., 1993, Pisegna and Wank, 1996). We recently identified a novel splice variant of the PAC1 receptor, PAC1 short (PAC1s), which binds not only PACAP38 and PACAP27 but also VIP with high affinity (Dautzenberg et al., 1999b). This splice variant and PAC1n, the normal PAC1 variant, are abundantly expressed in human brain, retina and Y-79 retinoblastoma cells (Dautzenberg et al., 1999b). In addition to signaling through Gs (Spengler et al., 1993, Pisegna and Wank, 1996), PAC1, VPAC1 and VPAC2 receptors also signal through the phospholipase C (PLC) pathway (Spengler et al., 1993, Pisegna and Wank, 1996).
Agonist-activated Gs-coupled GPCRs signal by stimulating the adenylyl cyclase-mediated conversion of ATP into cyclic AMP (cAMP), the intracellular second messenger. However, in the continuing presence of high concentrations of agonist, GPCR signaling is terminated rapidly via a process termed homologous desensitization (reviewed in Carman and Benovic, 1998, Ferguson et al., 1996, Post et al., 1999, Penn and Benovic, 1998, Pitcher et al., 1998, Koch et al., 2000). Homologous desensitization begins with the selective recruitment of a G-protein-coupled receptor kinase (GRK) that phosphorylates serine and/or threonine residues in the agonist-activated GPCR's cytoplasmic tail and/or third intracellular loop. Next, β-arrestin binds to the phosphorylated GPCR, decreasing the receptor's affinity for its cognate heterotrimeric G protein, and thereby uncoupling it from the Gα subunit by steric hindrance. To date, six GRKs have been cloned and sequenced: GRK1 (rhodopsin kinase), GRK2 (β-adrenergic receptor kinase or βARK1), GRK3 (βARK2), GRK4, GRK5, and GRK6 (Carman and Benovic, 1998, Ferguson et al., 1996, Penn and Benovic, 1998, Pitcher et al., 1998, Stoffel et al., 1997). Overexpression of GRK2 in various types of cell transfection systems has revealed that this kinase significantly augments the homologous desensitization of a number of GPCRs, including the β2-adrenergic receptor (Ferguson et al., 1996, Penn and Benovic, 1998, Pippig et al., 1993, Pitcher et al., 1998). For example, overexpression of GRK2, but not GRK5, was found to augment the homologous desensitization of endothelin receptors in HEK293 cells (Freedman et al., 1997). These and other studies suggest that GRKs selectively phosphorylate GPCRs (Koch et al., 2000, Penn and Benovic, 1998, Pitcher et al., 1998).
The highly localized and concentrated presence of GRK2 and GRK3 in brain synapses that receive neurotransmitters and neuropeptides released by presynaptic neurons suggests that homologous GPCR desensitization mediated by these kinases plays an important, albeit poorly understood, role in the regulation of neuronal signaling (Arriza et al., 1992). Since little is known about the molecular mechanisms regulating PAC1 receptor signaling in a physiological setting, we chose to investigate PAC1 receptor desensitization in human retinoblastoma Y-79 cells, which endogenously express high-affinity PAC1 receptors coupled via Gs to adenylyl cyclase (Olianas et al., 1996, Dautzenberg et al., 1999b). The primary goal of the present study was to characterize the time- and concentration-dependent parameters of GRK3-mediated homologous desensitization of retinoblastoma PAC1 receptors. Secondarily, we aimed to elucidate the roles of GRK3, protein kinase A (PKA), and protein kinase C (PKC) in the homologous and/or heterologous desensitization of retinoblastoma PAC1 receptors.
Section snippets
Materials, peptides and reagents
All cell culture reagents were purchased from Gibco/BRL (Eggenstein, Germany) except for aprotinin (Trasylol; Roche Diagnostics, Mannheim, Germany). Bovine serum albumin (BSA, fraction V) was obtained from Sigma (Munich, Germany). PACAP38 and PACAP27 (ovine) and VIP (human, porcine, rat) were purchased from Calbiochem (Bad Soden, Germany). Forskolin, H-89, dibutyryl-cyclic AMP (dbcAMP), staurosporine, and bisindolylmaleimide (BIS) were purchased from Calbiochem. The RT–PCR primers used in this
PACAP38-, PACAP27-, and VIP-stimulated cAMP accumulation in retinoblastoma cells
A comparison of cAMP accumulation in Y-79 cells pretreated with PACAP38, PACAP27 or VIP was performed. Incubation of Y-79 cells with picomolar concentrations of either of the two PACAP peptides robustly increased intracellular cAMP accumulation to a maximum (50 pmol/106 cells) that exceeded basal levels 100-fold (0.5 pmol/106 cells) (Fig. 1). However, PACAP38 (EC50=24±3 pM) was fourfold more efficacious in stimulating cAMP accumulation than PACAP27 (EC50=99±8 pM). In contrast, VIP was 1000-fold
Discussion
This study establishes that neural PAC1 receptors expressed in human retinoblastoma Y-79 cells undergo rapid, homologous desensitization following exposure to nanomolar levels of PACAP38 (t1/2 58 min) or PACAP27 (t1/2 164 min). During PAC1 receptor desensitization, EC50 values for PACAP-stimulated cAMP accumulation shifted rightward 5.3- and 2.1-fold in Y-79 cells pretreated with PACAP38 or PACAP27, respectively. It is important to note that PACAP38 homologously desensitized retinoblastoma PAC1
Acknowledgements
R.L.H. was supported by a VA Merit Review grant, the VA Mental Illness Research, Education and Clinical Center (MIRECC) of VISN22, and the NIMH Mental Health Clinical Research Center (PHS MH20914-14). The authors thank Sandra Braun and Sandra Wille for excellent technical assistance, and Dr R. Lefkowitz and Carl Stone who kindly provided the mouse monoclonal C5/1 antibody, GRK2 and GRK3 protein standards, the Western immunoblotting protocol, and invaluable advice. In addition, we thank Dr Heiko
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