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Beta-adrenergic receptor kinase-like activity and beta-arrestin are expressed in osteoblastic cells
M Bliziotes1, J Murtagh, K Wiren
1Bone and Mineral Research Unit, Portland VAMC, Oregon, USA.
Abstract:
Biologic responses to peptide calciotropic hormones, such as parathyroid hormone (PTH) and calcitonin, exhibit desensitization. As with most hormones, however, the mechanisms of desensitization are not completely understood. For the beta 2-adrenergic receptor (beta 2AR) system, which is coupled to adenylyl cyclase via the stimulatory guanine nucleotide-binding regulatory (G5) protein, homologous desensitization is mediated in part by a receptor-specific kinase (beta ARK) and a soluble cofactor (beta-arrestin). Recently, this system has been reported to be involved in rapid homologous desensitization of the PTH/parathyroid hormone receptor protein (PTHrP) receptor. We have identified the presence of this system in bone using reverse-transcriptase PCR. Nucleotide sequence of PCR fragments from ROS 17/2.8 cells revealed 100% identity with rat brain beta ARK1 and beta-arrestin 1 sequences. Northern analyses with RNA from ROS 17/2.8, UMR 106-H5 cells, and primary cultures of nontransformed neonatal rat calvariae demonstrated two mRNA species of 4 and 2.6 kilobases (kb) for beta ARK and 7.5 kb for beta-arrestin, comparable to those found in bovine brain. beta ARK-like activity was demonstrated in cytosolic extracts of the UMR 106-H5 cells by assessing phosphorylation of the retinal photoreceptor, rhodopsin, by the extracts. Phosphorylation was enhanced with light-activated rhodopsin and by bovine brain G beta gamma subunits; heparin inhibited phosphorylation. These findings are characteristic of beta ARK. Expression of beta-arrestin in the UMR 106-H5 cells was confirmed by immunoblot. Thus, osteoblastic cells express proteins, beta ARK, and beta-arrestin, which may regulate desensitization of calciotropic hormone receptors.
Insights
Osteoblastic cells express beta-adrenergic receptor kinase (beta ARK) and beta-arrestin, proteins crucial for desensitization. These findings suggest a mechanism for regulating calciotropic hormone receptor responses in bone.
Area of Science:
- Molecular biology
- Endocrinology
- Cell biology
Background:
- Biologic responses to calciotropic hormones like parathyroid hormone (PTH) involve desensitization, but mechanisms remain unclear.
- The beta 2-adrenergic receptor (beta 2AR) system, involving beta-adrenergic receptor kinase (beta ARK) and beta-arrestin, mediates homologous desensitization.
- This system has been implicated in the rapid desensitization of the PTH/parathyroid hormone receptor protein (PTHrP) receptor.
Purpose of the Study:
- To investigate the presence and role of the beta ARK/beta-arrestin system in bone cells.
- To explore the potential involvement of these proteins in the desensitization of calciotropic hormone receptors.
Main Methods:
- Reverse-transcriptase PCR (RT-PCR) to detect gene expression in osteoblastic cells (ROS 17/2.8, UMR 106-H5) and neonatal rat calvariae.
- Nucleotide sequencing of PCR fragments to confirm identity.
- Northern blot analysis to determine mRNA sizes for beta ARK and beta-arrestin.
- Assay of beta ARK-like activity using rhodopsin phosphorylation in cytosolic extracts.
- Immunoblot analysis to confirm beta-arrestin expression.
Main Results:
- RT-PCR and sequencing confirmed the presence of beta ARK1 and beta-arrestin 1 in ROS 17/2.8 cells, identical to rat brain sequences.
- Northern analysis revealed mRNA species for beta ARK (4 and 2.6 kb) and beta-arrestin (7.5 kb) in osteoblastic cells and calvariae, comparable to bovine brain.
- Cytosolic extracts from UMR 106-H5 cells exhibited beta ARK-like activity, demonstrated by light-activated rhodopsin phosphorylation, enhanced by G beta gamma subunits and inhibited by heparin.
- Immunoblotting confirmed beta-arrestin expression in UMR 106-H5 cells.
Conclusions:
- Osteoblastic cells express beta ARK and beta-arrestin proteins.
- These proteins are likely involved in the homologous desensitization of calciotropic hormone receptors in bone.
- This study identifies a key molecular mechanism regulating bone cell responses to hormonal signals.