Direct mapping of the interface between parathyroid hormone (PTH) and its receptor (hPTH1-Rc Parathyroid hormone (PTH)1 is the major regulator of calcium levels in blood and plays a role in the regulation of bone remodeling (1). Given intermittently, PTH displays anabolic activity in bone and, therefore, has considerable therapeutic potential (2). PTH and PTH-related protein exert their actions via a seven-transmembrane (TM) domain-containing receptor (PTH1-Rc) (3) belonging to a subfamily of related G proteincoupled receptors (4 -11). The PTH1-Rc is coupled to both adenylyl cyclase/cyclic AMP and phospholipase C/inositol 1,4,5-trisphosphate/cytosolic calcium intracellular signaling pathways (12)(13)(14)(15).Understanding the molecular mechanism of ligand recognition and signal transduction by the PTH1-Rc may identify new directions for the design of novel hormone analogs for the treatment of diseases such as osteoporosis, hypercalcemia of malignancy and hyperparathyroidism (16). In order to directly identify the structural elements involved in PTH-PTH1-Rc interactions, we employed a photoaffinity scanning approach (17). The generation of covalently linked ligand-receptor conjugates and the identification of the cross-linked domains allows mapping of the interface between hormone and receptor. Photoaffinity cross-linking has been successfully applied in defining interactions between small peptides, such as substance P (18 -20), cholecystokinin (21), and vasopressin (22), and their receptors. Recently, we used this general approach to identify directly the interaction between position 13 of PTH and a 17-amino acid domain (residues 173-189) of the hPTH1-Rc (17).We now report the evaluation of a series of photoreactive analogs obtained by a "p-benzoylphenylalanine (Bpa) scan" of the principal receptor activation domain (residues 1-6) of 34)), maintained full potency and led to the identification of a second "contact domain" between PTH and hPTH1-Rc. This information allows us to create, for the first time, a model describing interactions of hPTH-(1-34) with its receptor based on direct identification of the interacting regions. EXPERIMENTAL PROCEDURESMaterials-Boc-protected amino acids, N-hydroxybenzotriazole, N,NЈ-dicyclohexylcarbodiimide, and p-methylbenzydrylamine resin were purchased from Applied Biosystems (Foster City, CA). Boc-(3-iodo)tyrosine ] was from Peninsula Laboratories (Belmont, CA). B&J brand dichloromethane, N-methylpyrrolidone, and acetonitrile were obtained from Baxter (McGraw Park, IL). IODOGEN ® and 2-(2Ј-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole) were purchased from Pierce. Cyanogen bromide was from Aldrich. Na

Multiple genetic alterations occur in melanoma, a lethal skin malignancy of increasing incidence1 ,2 . These include mutations that activate Ras and two of its effector cascades, Raf and phosphoinositide 3-kinase (PI3K). Ras and Raf induction can occur via active N-Ras and B-Raf mutants as well as by gene amplification 3-5. Activation of PI3K pathway components occurs by PTEN loss and by AKT amplification 6-8 . Melanomas also commonly display impairment of p16 INK4A -CDK4-Rb and ARF-HDM2-p53 tumor suppressor pathways. CDKN2A mutations can produce p16 INK4A and ARF protein loss 5,9-11 . Rb bypass can also occur through activating CDK4 mutations as well as by CDK4 amplification 5,12 . In addition to ARF deletion, p53 pathway disruption can result from dominant-negative TP53 mutations 5,13 . Other findings in melanoma include hTERT amplification 5 . The ability of any of these mutations to induce human melanocytic neoplasia, however, is unknown. The present work characterizes pathways sufficient to generate human melanocytic neoplasia and demonstrates that genetically altered human tissue facilitates functional analysis of mutations observed in human tumors.

Melanoma is a cancer of the neural crest-derived cells that provide pigmentation to skin and other tissues. Over the past 4 decades, the incidence of melanoma has increased more rapidly than that of any other malignancy in the United States. No current treatments substantially enhance patient survival once metastasis has occurred. This review focuses on recent insights into melanoma genetics and new therapeutic approaches being developed based on these advances.

Parathyroid hormone (PTH) regulates calcium and phosphate metabolism through a G-protein-coupled receptor which is shared with PTH-related protein (PTHrP). Therefore, structure-activity studies of PTH and PTHrP with their common receptor provide an unusual opportunity to examine the structural elements in the two hormones and their common receptor which are involved in the expression of biological activity. Our approach to studying the nature of the bimolecular interface between hormone and receptor is to use a series of specially designed photoreactive benzophenone- (BP-) containing PTH analogs in "photoaffinity scanning" of the PTH/PTHrP receptor. In this report we describe a series of BP-containing agonists and antagonists which have been synthesized by solid-phase methodology and characterized physiocochemically and biologically. Each of the 12 analogs contains a single BP moiety at a different defined position. Examples of BP-containing agonists prepared and studied in human osteogenic sarcoma Saos-2/B-10 cells are [Nle8,18,Lys13(epsilon-pBZ2),L-2-Nal23,Tyr34]bPTH(1-34 )NH2(K13)(Kb = 13 nM; Km = 2.7 nM) and [Nle8,18,L-Bpa23,Tyr34[bPTH(1-34)NH2(L-Bpa23) (Kb = 42 nM; Km = 8.5 nM). Another BP-containing analog, [Nle8,18,D-2-Nal12,Lys13(epsilon-pBZ2),L-2-Nal23 ,Tyr34]bPTH(7-34)NH2, was a potent antagonist (Kb = 95 nM; Ki = 72 nM). The amino acids substituted by residues carrying the BP moiety span the biologically active domain of the hormone (Phe7, Gly12, Lys13, Trp23, and Lys26). Analysis of photo-cross-linked conjugates of PTH/PTHrP receptor with BP-containing PTH analogs should help to identify the "contact points" between ligand and receptor.