Norman D. Boyd - US grants

In addition to their ability to convert chemical signals into electrical responses, neurotransmitter-gated ion channels, they are dynamic molecular entities, subject to regulation of their responsiveness over a wide temporal range. The focus of the experiments described in this proposal is on the mechanisms that regulate the neural acetylcholine receptor (AcChR) of PC12 cells, a clonal sympathetic cell line. The kinetic properties of agonist- mediated desensitization will be examined by sodium influx measurements of receptor activity. The possibility that phosphorylation-dephosphorylation reactions are involved in the development of long lasting desensitization that is characterized by slow onset and recovery rates, will be investigated by obtaining measurements, in parallel, of the extent of AcChR phosphorylation. A binding assay to quantitate nAcChRs of PC12 cells will be developed to examine whether deactivation, an agonist-induced process that produces an irreversible dimunution of the permeability response, involves a rapid loss of surface receptors. The relationship between deactivation and the receptor states involved in desensitization will be examined by kinetic analysis of the effects on the rates of each of these processes by different cholinergic antagonists. The role of other synaptic components both extracellular, e.g. substance P, and intracellular, e.g. Ca2+ and cAMP, in modulating further these agonist-mediated processes will also be evaluated by the same biochemical techniques. By examining the mechanisms of short- and long-term regulation of nAcChR by agonists and other synaptic components, the proposed studies will aid in evaluating the possible role of postsynaptic mechanisms in modulating synaptic efficacy. A better understanding of these processes will shed light on one of the most remarkable properties of the brain: its ability to be regulated by experience.

Acetylcholine, norepinephrine and substance P control salivary secretion by binding to receptors on the surface of salivary acinar cells to produce specific intracellular signals. Two signal transduction pathways have been identified: one involves cAMP synthesis and the other involves phosphoinositide hydrolysis and Ca2+ mobilization. The cAMP pathway, including the role of G proteins as transducers, is well understood. Less is known about the phosphoinositide /Ca2+ pathway although there is evidence that a G protein is also involved in this pathway. The identity of the G protein is unknown; its lack of sensitivity to cholera and pertussis toxins would seem to rule out the involvement of any of the G proteins that have been purified to date. The objectives of this proposal are to identify this novel G protein, provisionally termed G(p), and to characterize its biochemical and functional properties. The specific aims are: 1) isolation and biochemical characterization of a pertussis toxin-insensitive G protein(s) from submaxillary glands that can functionally interact with the SP receptor; 2) identification of the G protein associated with the SP receptor, by chemical crosslinking; 3) analysis of the functional interactions of purified G with the SP receptor and phospholipase C; 4) partial amino acid sequence analysis of homogeneous GP. Two new experimental approaches will be used. Salivary gland membranes, depleted of their endogenous G proteins by treatment with alkaline buffer, will be reconstituted with active G proteins obtained during chromatographic fractionation of G proteins from horse submaxillary gland. This convenient and sensitive assay detects G proteins that can functionally interact with SP receptors to restore GTP-sensitive, high affinity agonist binding The second approach will involve the use of a novel photoreactive analog of SP. (125)I-labelled Phe8(pBz)-SP can be photoincorporated efficiently into the G -coupled SP receptor. Gp will then be chemically crosslinked to the photolabelled SP receptor and the resulting radiolabelled covalent adduct subjected to compositional analysis by SDS-PAGE and immunodetection techniques. There are disease states that result from altered G protein function. Understanding the role of G in the phosphoinositide/Ca2+ pathway will permit an assessment of altered transduction in certain states of salivary gland dysfunction.

DESCRIPTION (adapted from the applicant's abstract): The three- dimensional structure of the binding pockets of the SP and SK receptors will be defined with respect to their transmembrane alpha-helices and extracellular domains by determining the sites of covalent attachment of a series of SP and SK derivatives containing the photoreactive amino acid p-benzoylphenylalanine spaced at different positions along the amino acid sequences of the two peptides. For each photoaffinity ligand, the amino acid residue of the binding site that becomes covalently labelled will be determined by microsequencing of purified receptor fragments initially located in the primary sequence with the aid of site-specific antibodies and mass spectrometry. A comparative analysis of the similar and distinctive features of these binding domains should provide insight into the molecular basis of peptide selectivity and receptor activation. A related project will be to determine the site of attachment of a tritiated photoreactive azide derivative of the nonpeptide SP antagonist CP-96,345. An understanding of the relationship between the sites for peptide agonists and the nonpeptide antagonist may provide insight into the mechanism of the antagonist effects of this compound, and the information to be gained from the project as a whole may advance the rational design of therapeutic agents for clinical entities in which these peptides participate.

Electrospray (ESI) and matrix-assisted laser desorption/ionization (NLALDI) mass spectrometry, in combination with proteolyic digestion and BPLC separation, have been used to verify the cDNA-predicted amino acid sequence of chicken type II collagen. The type II collagen molecule is composed of three identical polypeptide chains [alpha, (11) chains] intertwined in a triple helix. The single triple helical domain comprises about 96% of the amino acid sequence and consists of repeating tripeptides of the form glycine-X-Y (G-X-Y). The helical domain is flanked on both the N-terminal and C-terminal ends by short non-helical domains known as telopeptides. Themolecular weight for the unmodified chicken type 11 collagen triple helix predicted from the cDNA sequence is 294 kDa. Posttranslational modifications occur at numerous sites; the best studied are hydroxylation of prolines and lysines in the Y position of the G-X-Y sequence, glycosylation of hydroxylated lysines, and interhelical cross-linkages between lysines and hydroxylysines. Mass spectrometric analysis of this protein is very challenging because several factors produce heterogeneity. Incomplete modification at the large number of potential sites for post-translational modifications produces a variety of forms. During the solubilization of type 11 collagen from chicken sterna by pepsin digestion, the C-terminal telopeptide may be cleaved at several different points, producing additicrnal heterogeneity. The digestion may also generate "tags" from the crosslinked peptides that are attached to the main chain. Chicken type 11 collagen hasbeen heat denatured and digested with endoproteinase Lys-C. The Lys-C peptides were separated by reversed-phase FIPLC, collected and examined by MALDI-TOF MS and ESI MS. A total of 131 different peptides with molecular weights ranging from 711 Da to 14,027 Da have been identified to date. MS/MS analyses and secondary digestions have been performed to determine peptide sequence and to locate modification sites within a peptide. Our mass spectrometric analysis has identified numerous sites of hydroxylation and glycosylation and has identified several C-termini. This analysis has enabled us to confirm most of the residues predicted from the cDNA sequence and to correct a few misassignments The results show a high degree of heterogeneity in hydroxylation and glycosylation with attendant shifts in chromatographic behavior.

This renewal is a continuation of studies on the structure and function of the substance P (SP) receptor. The long term objective of this proposal is to understand, at the molecular level, the basis of agonist and antagonist specificity, receptor activation and downstream signaling events. Our approach will be to use photoreactive SP analogs containing p-benzoylphenylalanine substituted at different positions in the eleven amino acid sequence of SP. The site(s) of covalent attachment of these photoligands on the SP receptor will be determined by MALDI-mass spectrometric analysis of isolated photolabeled receptor fragments, which are generated by enzymatic and/or chemical cleavage of the receptor. These studies will aid in defining the SP binding pocket and orient the peptide within that pocket. We will develop benzophenone-containing derivatives of non-peptide SP antagonists as photoprobes of the antagonist binding domain(s). An understanding of the relationship between the binding sites for peptide agonist and the non-peptide antagonists forms the foundation for understanding underlying mechanisms of antagonism, which is essential for drug development. The availability of photoreactive peptide agonists, together with chemical crosslinking and immunodetection, provides us with tools to identify specific G proteins and other regulatory proteins which interact with the SP receptor. This approach will provide important information about the signal transduction machinery associated with the SP receptor. As it has been estimated that up to 60 percent of all medicines used today exert their effects through G protein signaling pathways, these studies may provide new and interesting information of basic clinical relevance. The peptide substance P has attracted considerable attention because of its multiple biological activities: as a neurotransmitter in the central, sensory and autonomic nervous systems; as an agent that causes contraction of smooth muscle in the gastrointestinal tract and as a mediator of inflammation and immune responses. SP has been implicated in a number of sensory and neurogenerative disorders. The information provided by our continuing effort should form the basis for understanding and development of novel SP receptor agonists and antagonists.