The type I PTH/PTH-related peptide receptor (PTH1R), 2 a seventransmembrane receptor (7TMR) highly expressed in the kidney and bone, plays a fundamental role in the regulation of calcium homeostasis, as well as in bone formation and resorption. Ligands for PTH1R including PTHrp and PTH are involved in the etiology and treatment of disease processes such as hypercalcemia of malignancy and osteoporosis. The actions of PTH, however, are complex. PTH is known for both anabolic and catabolic effects on bone, which are dependent upon intermittent or persistent exposure, respectively (1-3). The mechanistic basis of these effects on bone remodeling are not well understood.The intracellular signaling pathways activated by PTH and PTHrP via the PTH1R receptor include G s -mediated activation of adenylate cyclase, resulting in cAMP production and PKA activation, and G q/11 -mediated PLC␤ stimulation, leading to inositol 1,4,5-trisphosphate (IP 3 ) production, calcium mobilization, and PKC activation (4 -7). It has also been demonstrated that PTH activates the Raf-MEK-ERK MAP kinase (MAPK) cascade through both PKA and PKC in a cell-specific and G protein-dependent manner (8 -10). MAPKs activated in response to stimulation by many different classes of cell surface receptors, including growth factor receptor tyrosine kinases and 7TMRs, regulate cell growth, division, differentiation, and apoptosis (11). PTHstimulated activation of MAPK is known to have proliferative effects in kidney and bone (12, 13).There is growing evidence for novel 7TMR signaling mechanisms that are distinct from the classical G protein second messenger-dependent pathways. One such mechanism involves ␤-arrestins, a small family of cytosolic proteins initially identified for their central role in 7TMR desensitization. ␤-Arrestins are recruited to agonist-occupied 7TMRs that have been phosphorylated by specialized G protein-coupled receptor kinases (GRKs) and sterically inhibit receptor-G protein coupling resulting in homologous receptor desensitization. Additionally, ␤-arrestins act as adaptors in clathrin-mediated receptor endocytosis (14, 15). The role of ␤-arrestins acting as signal transducers through the formation of scaffolding complexes with accessory effector molecules such as Src, Ras, ERK1/2, JNK3, and MAPK kinase 4 (MKK4) is becoming increasingly recognized (16 -20).The potential signaling diversity of 7TMRs suggests the possible existence of multiple discrete "active" receptor conformations. This implies that specific ligands might direct distinct signaling responses by preferentially stabilizing one or more of these active conformations. In the simple two-state model of receptor activation, agonists are defined as drugs that stabilize the active receptor conformation, which in turn promotes G protein activation. Conversely, an inverse agonist preferentially binds to the inactive receptor conformational state thereby reduc-

Formation of a ternary complex between human MutS␣, MutL␣, and heteroduplex DNA has been demonstrated by surface plasmon resonance spectroscopy and electrophoretic gel shift methods. Formation of the hMutL␣⅐hMutS␣⅐heteroduplex complex requires a mismatch and ATP hydrolysis, and depends on DNA chain length. Ternary complex formation was supported by a 200-base pair G-T heteroduplex, a 100-base pair substrate was somewhat less effective, and a 41-base pair heteroduplex was inactive. As judged by surface plasmon resonance spectroscopy, ternary complexes produced with the 200-base pair G-T DNA contained ϳ0.8 mol of hMutL␣/mol of heteroduplex-bound hMutS␣. Although the steady-state levels of the hMutL␣⅐hMutS␣⅐ heteroduplex were substantial, this complex was found to turn over, as judged by surface plasmon resonance spectroscopy and electrophoretic gel shift analysis. With the former method, the majority of the complexes dissociated rapidly upon termination of protein flow, and dissociation occurred in the latter case upon challenge with competitor DNA. However, ternary complex dissociation as monitored by gel shift assay was prevented if both ends of the heteroduplex were physically blocked with streptavidin⅐biotin complexes. This observation suggests that, like hMutS␣, the hMutL␣⅐hMutS␣ complex can migrate along the helix contour to dissociate at DNA ends.The MutS and MutL homologs, which are required for the initiation of mismatch repair, have been implicated in the correction of DNA biosynthetic errors, the transcription-coupled repair of DNA damage, and the fidelity of genetic recombination (1-6). In mammalian cells, MutS␣ (MSH2⅐MSH6 heterodimer), MutS␤ (MSH2⅐MSH3 heterodimer), and MutL␣ (MLH1⅐PMS2 heterodimer) are also thought to function as lesion sensors for certain types of DNA damage that kill by activating apoptosis (2, 6, 7).Repair in the Escherichia coli system is initiated by the binding of MutS to a mismatch (8 -10). Formation of a MutL⅐MutS⅐heteroduplex complex has been demonstrated by DNase I footprint analysis (11), electron microscopy (12), and surface plasmon resonance spectroscopy (SPRS) 1 (13), with assembly of this ternary complex being ATP-dependent. Several lines of evidence indicate that assembly of the ternary complex is required for subsequent steps in mismatch repair. Both MutS and MutL are required for the mismatch-dependent activation of the d(GATC) endonuclease activity of MutH, which cleaves the unmethylated strand of a hemimethylated d(GATC) site, with the ensuing strand break serving to direct repair to the unmethylated DNA strand (14). MutS and MutL are also required for the mismatch-dependent activation of DNA helicase II, which enters the helix at the strand break and initiates the excision step of repair (15). Formation of a MutL␣⅐MutS␣⅐heteroduplex complex has been demonstrated by electrophoretic gel shift analysis with yeast mismatch repair proteins using synthetic heteroduplexes of ϳ50 base pairs (bp) in size (16, 17). However, using gel shift methods and surface plasmon reson...