An intramolecular contact in Galpha transducin that participates in maintaining its intrinsic GDP release rate.

Receptor mediated stimulation of the G protein-alpha subunit leads to exchange of GDP for GTP, activating the protein. Spontaneous GDP release from Galpha can also lead to the active state, if GTP in solution binds the nucleotide binding pocket. The purpose of this study is to evaluate the molecular determinants for maintaining the spontaneous GDP release rates between two Galpha subunits. Galpha(t) has a low rate of nucleotide release, compared to Galpha(i1). Galpha(t/i1) chimeras were used to explore the molecular basis for this behavior. The C-terminal alpha4-helix, the N-terminal 56 residues and the Switch I/II regions of Galpha(t) were shown to affect the low spontaneous GDP release rate in Galpha(t). A specific molecular contact between Asp26 and Asn191 was found in Galpha(t) that is not present in Galpha(i1). In two chimeras disrupting this interaction produced an increased spontaneous GDP release; restoring the contact present in Galpha(t) into these chimeras decreased the GDP release rate by half as compared to the original chimeras. Similarly, introduction of this contact in wild-type Galpha(i1) decreased the GDP release rate of Galpha(i1) by half. Differences in GDP release rates may reflect physiological roles these proteins play in living systems.

G alpha minigenes expressing C-terminal peptides serve as specific inhibitors of thrombin-mediated endothelial activation.

The C termini of G protein alpha subunits are critical for binding to their cognate receptors, and peptides corresponding to the C terminus can serve as competitive inhibitors of G protein-coupled receptor-G protein interactions. This interface is quite specific as a single amino acid difference annuls the ability of a G alpha(i) peptide to bind the A(1) adenosine receptor (Gilchrist, A., Mazzoni, M., Dineen, B., Dice, A., Linden, J., Dunwiddie, T., and Hamm, H. E. (1998 ) J. Biol. Chem. 273, 14912--14919). Recently, we demonstrated that a plasmid minigene vector encoding the C-terminal sequence of G alpha(i) could specifically inhibit downstream responses to agonist stimulation of the muscarinic M(2) receptor (Gilchrist, A., Bunemann, M., Li, A., Hosey, M. M., and H. E. Hamm (1999) J. Biol. Chem. 274, 6610--6616). To selectively antagonize G protein signal transduction events and determine which G protein underlies a given thrombin-induced response, we generated minigene vectors that encode the C-terminal sequence for each family of G alpha subunits. Minigene vectors expressing G alpha C-terminal peptides (G alpha(i), G alpha(q), G alpha(12), and G alpha(13)) or the control minigene vector, which expresses the G alpha(i) peptide in random order (G(iR)), were systematically introduced into a human microvascular endothelial cell line. The C-terminal peptides serve as competitive inhibitors presumably by blocking the site on the G protein-coupled receptor that normally binds the G protein. Our results not only confirm that each G protein can control certain signaling events, they emphasize the specificity of the G protein-coupled receptor-G protein interface. In addition, the C-terminal G alpha minigenes appear to be a powerful tool for dissecting out the G protein that mediates a given physiological function following thrombin activation.

Tissue-specific expression of a Ca(2+)-activated K+ channel is controlled by multiple upstream regulatory elements.

The electrical properties of a cell are produced by the complement of ion channels that it expresses. To understand how ion-channel gene expression is regulated, we are studying the tissue-specific regulation of the slowpoke (slo) Ca(2+)-activated K+ channel gene. This gene is expressed in the central and peripheral nervous system, in midgut and tracheal cells, and in the musculature of Drosophila melanogaster. The entire transcriptional control region has been cloned previously and shown to reproduce the tissue and developmental expression pattern of the endogenous gene. Here we demonstrate that s/o has at least four promoters distributed over approximately 4.5 kb of DNA. Promoter C1 and C1c display a TATA box-like sequence at the appropriate distance from the transcription start site. Promoters C1b and C2, however, are TATA-less promoters. C1, C1b, and C1c transcripts differ in their leader sequence but share a common translation start site. C2 transcripts incorporate a new translation start site that appends 17 amino acids to the N terminus of the encoded protein. Deletion analysis was used to identify sequences important for tissue-specific expression. We used a transgenic in vivo expression system in which all tissues and developmental stages can be assayed easily. Six nested deletions were transformed into Drosophila, and the expression pattern was determined using a lacZ reporter in both dissected tissues and sectioned animals. We have identified different sequences required for expression in the CNS, midgut, tracheal cells, and muscle.