This study investigates the poor reversibility of salmon calcitonin (sCT) binding to rat and human calcitonin receptors. Efficacy of CT and analogue peptides in 125 IsCT binding competition and cAMP assays was compared with the dissociation kinetics of 125 I-labelled peptides. Assessment was performed on cells stably expressing either rat or human calcitonin receptors. Dissociation kinetics of the antagonists, sCT(8-32) and AC512, revealed that binding was rapidly and completely reversible at the receptors, despite high affinity binding, suggesting that poor reversibility required the active conformation of the receptor. G protein coupling was not essential as the dissociation kinetics of 125 I-sCT binding to cell membranes did not significantly alter in the presence of GTP S. Time course experiments established that the transition to irreversibility was slow, while the reversible component of binding appeared to involve a single population of either receptor states or binding sites. Pre-bound 125 I-human CT dissociated rapidly from the receptors, indicating that not all agonists bound irreversibly. To identify structural features of sCT that contribute to its poor reversibility, dissociation kinetics of sCT analogues with various structural modifications were examined. Increasing truncation of N-terminal residues of sCT analogues led to a corresponding increase in the rate of peptide dissociation. Salmon CT peptides which had been substituted at the N-terminus by 13-21 residues of human CT (hCT) were equipotent with sCT in binding competition and cAMP accumulation assays but exhibited a dissociation rate similar to hCT. In contrast, despite lower affinity and efficacy at the receptors, the chimeric analogue sCT(1-16)-hCT(17-32) displayed poorly reversible binding, similar to sCT. Analysis of the dissociation kinetics of sCT analogues with differing -helix forming potential indicated that the ability to form -helical secondary structure was an important factor in the rate of ligand dissociation. We hypothesise that poor reversibility results from a conformational change in the receptor and/or ligand and that this is dependent, at least in part, on interaction with residues constrained within the -helix of the peptide.

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The structure/function relationship of salmon calcitonin (sCT) analogues was investigated in heterologous calcitonin receptor (CTR) expression systems. sCT analogues with progressive amino-terminal truncations intermediate of sCT-(1-32) to sCT-(8-32) were examined for their ability to act as agonists, antagonists, or inverse agonists. Two CTR cell clones, B8-H10 and G12-E12, which express approximately 5 million and 25,000 C1b receptors/cell, respectively, were used for this study. The B8-H10 clone has an approximately 80-fold increase in basal levels of intracellular cAMP due to constitutive activation of the overexpressed receptor. In whole-cell competition binding studies, sCT-(1-32) was more potent than any of its amino-terminally truncated analogues in competition for 125I-sCT binding. In cAMP accumulation studies, sCT-(1-32) and modified analogues sCT-(2-32) and sCT-(3-32) had agonist activities. SDZ-216-710, with an amino-terminal truncation of four amino acids, behaved as a partial agonist/antagonist, whereas amino-terminal truncations of six or seven amino acid residues produced a 16-fold reduction in basal cAMP levels and attenuated the response to the agonist sCT-(1-32) in the constitutively active CTR system. This inverse agonist effect was insensitive to pertussis toxin inhibition. In contrast, the inverse agonist activity of these peptides was not observed in the nonconstitutively active CTR system, in which sCT analogues with amino-terminal truncations of four or more amino acids behaved as neutral competitive antagonists. These results suggest that the inverse agonist activity is mediated by stabilization of the inactive state of the receptor, which does not couple to G protein, and attenuates basal signaling initiated by ligand-independent activation of the effector adenylyl cyclase.

Despite the fact that inositol hexakisphosphate (InsP 6 ) is the most abundant inositol metabolite in cells, its cellular function has remained an enigma. In the present study, we present the first evidence of a protein kinase identified in rat cerebral cortex/hippocampus that is activated by InsP 6 . The substrate for the InsP 6 -regulated protein kinase was found to be the synaptic vesicle-associated protein, pacsin/syndapin I. This brain-specific protein, which is highly enriched at nerve terminals, is proposed to act as a molecular link coupling components of the synaptic vesicle endocytic machinery to the cytoskeleton. We show here that the association between pacsin/syndapin I and dynamin I can be increased by InsP 6 -dependent phosphorylation of pacsin/syndapin I. These data provide a model by which InsP 6 -dependent phosphorylation regulates synaptic vesicle recycling by increasing the interaction between endocytic proteins at the synapse.A large number of G-protein-coupled and growth factor receptors activate phospholipase C to stimulate the hydrolysis of the phospholipid, phosphatidylinositol 4,5-bisphosphate, producing inositol 1,4,5-trisphosphate (InsP 3 ) 1 and diacylglycerol. InsP 3 has an established second messenger role in mobilizing intracellular calcium stores (1) and can be phosphorylated to inositol 1,3,4,5-tetrakisphosphate (InsP 4 ), which is proposed to play a role in capacitative calcium entry (2) and as an activator of ras-GAP activity (3). The sequential dephosphorylation of InsP 3 and InsP 4 gives rise to a host of inositol phosphate isomers in a cyclic metabolic pathway that results in inositol returning to the phosphoinositide pool (4).In contrast to this well understood signaling pathway the role of inositol hexakisphosphate (InsP 6 ), the most abundant cellular inositol metabolite, is very poorly understood. Present at concentrations 10 -100 times greater than InsP 3 (15-60 M) (5, 6), InsP 6 has long been thought of as metabolically rather inert with levels that do not change in response to cell surface receptor activation. However, the discovery of higher phosphorylated forms of InsP 6 has led to the realization that the InsP 6 pool is rapidly turning over in a continual cycle of phosphorylation and dephosphorylation (7,8). Furthermore, a role for InsP 6 in synaptic vesicle trafficking has emerged from studies showing that InsP 6 binds with high affinity to a number of proteins that are involved in exo/endocytosis including the clathrin assembly proteins AP2 and AP3 and the synaptic vesicle calcium-sensing protein synaptotagmin (9 -12).In the present study, we have investigated the possibility of the existence of a protein kinase that is regulated by InsP 6 . We found that the pacsin/syndapin I, a synaptic vesicle-associated protein that acts as a molecular link coupling the endocytic machinery to the cytoskeleton, is phosphorylated by a protein kinase that is regulated by InsP 6 . In addition, InsP 6 -regulated phosphorylation of pacsin/syndapin I increases the interaction betwe...

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