No abstract

The ␤-adrenergic receptor kinase (␤ARK) is a member of growing family of G protein coupled receptor kinases (GRKs). ␤ARK and other members of the GRK family play a role in the mechanism of agonist-specific desensitization by virtue of their ability to phosphorylate G protein-coupled receptors in an agonist-dependent manner. ␤ARK activation is known to occur following the interaction of the kinase with the agonist-occupied form of the receptor substrate and heterotrimeric G protein ␤␥ subunits. Recently, lipid regulation of GRK2, GRK3, and GRK5 have also been described. Using a mixed micelle assay, GRK2 (␤ARK1) was found to require phospholipid in order to phosphorylate the ␤ 2 -adrenergic receptor. As determined with a nonreceptor peptide substrate of ␤ARK, catalytic activity of the kinase increased in the presence of phospholipid without a change in the K m for the peptide. The molecular mechanisms involved in signal transduction of G protein-coupled receptors are best understood in the visual system where rhodopsin serves as the "receptor" for light (1) and the ␤-adrenergic pathway in which the ␤-adrenergic receptor (␤AR) 1 binds catecholamines (2, 3). A feature common to both model systems as well as many other G protein receptors is the diminished responsiveness with time to a signal of equal intensity. This phenomenon is known as desensitization (4) and exhibits both an agonist-specific and nonspecific pattern. Rapid, agonist-specific desensitization of rhodopsin and the ␤ 2 -adrenergic receptor (␤ 2 AR) occurs in response to the phosphorylation of the receptor by the enzymes rhodopsin kinase and the ␤-adrenergic receptor kinase (␤ARK) (5). Rhodopsin kinase and ␤ARK are members of a family known as G proteincoupled receptor kinases (GRKs). A common feature to the GRK family of kinases is multi-site phosphorylation of receptor substrates in response to agonist occupancy (6). The relationship between agonist occupancy and receptor phosphorylation by GRKs is key to the specificity of the desensitization process, while other kinases such as protein kinase A and C play a role in nonspecific or heterologous desensitization. Two possible mechanisms could explain the enhanced phosphorylation of the activated form of the receptor by kinases of the GRK family. First, receptor occupancy may induce a conformational change exposing potential phosphorylation sites previously sequestered from the kinase. Alternatively, interaction of the kinase with the agonist-bound form of the receptor could result in enhanced catalytic activity of the kinase. The bulk of the experimental evidence supports the latter hypothesis (7-9). In addition to the enhanced catalytic activity of GRKs in the presence of agonist-occupied receptor, GRK2 and GRK3 activity is also increased by heterotrimeric G protein ␤␥ subunits (10 -13). The potential for finely controlled desensitization by the interplay of receptors and ␤␥ subunits is an exciting possibility given the evidence for dual regulation of GRK2 and GRK3 by these proteins (14). While G prot...

We developed a reproducible, relatively rapid bioassay that quantitatively correlates with the osteoinductive capacity of demineralized bone matrix obtained from human long bones. We have found that Saos human osteosarcoma cells proliferate in response to incubation with demineralized bone matrix and that an index of this proliferative activity correlates with demineralized bone matrix-induced osteogenesis in vivo. The bioassay (Saos cell proliferation) had an interassay coefficient of variation of 23 +/- 2% and an intra-assay coefficient of 11 +/- 1%. Cell proliferation was normalized to a standard sample of demineralized bone matrix with a clinically high osteoinductive capacity, which was assigned a value of one. The Saos cell proliferation for each sample was related to the standard and assigned a value placing it into the low (0.00-0.39), intermediate (0.40-0.69), or high (0.70-1.49) osteoinductive index group. Osteoinduction of human demineralized bone matrix was quantitated by expressing new bone formation as a function of the total bone volume (new bone plus the demineralized bone powder). The demineralized bone matrix was placed in pouches formed in the rectus abdominis muscles of athymic rats, and endochondral bone formation was assessed at 35 days following implantation, when marrow spaces in the ossicles were formed by new bone bridging the spaces between demineralized bone matrix particles. The proliferative index correlated with the area of new bone formation in histological sections of the newly formed ossicles. When the proliferative index (the osteoinductive index) was divided into low, intermediate, and high groups, the correlation between it and new bone formation (osteoinduction) was 0.850 (p < 0.0005) in 25 samples of demineralized bone matrix. There was no overlap in the osteoinduction stimulated between the samples with low and high osteoinductive indices. We conclude that the proliferation assay is useful for the routine screening of bone allograft donors for osteoinductive potential. Furthermore, the two-dimensional area of new bone formation, as it relates to total new bone area, is a quantitative measure of osteoinduction.