Alpha-helical distorting substitution disrupt coupling between m3 muscarinic receptor and G proteins.

Acetylcholine stimulation of the m3 or m2 muscarinic receptor expressed in Xenopus laevis oocytes induces either a fast transient or slowly oscillating calcium-sensitive chloride current. The speed of these currents reflects the efficiency of receptor coupling to guanine nucleotide-binding proteins and phosphatidylinositol (PI) turnover. Point mutations of the m3 receptor were made in a region of the third cytoplasmic loop to test whether receptor function relied on an alpha-helical structure of the G protein-coupling domain. Proline substitution for glutamate at position 257 disrupted the m3 response. Also, single alanine insertions between residues 259 and 260 disrupted the m3 receptor-stimulated response while double alanine insertions at this site had no effect. Based on these results, we suggest that a region of the third cytoplasmic loop of the m3 receptor possesses an amphipathic alpha-helical conformation.

Somatostatin stimulates BKCa channels in rat pituitary tumor cells through lipoxygenase metabolites of arachidonic acid.

The stimulation of large-conductance, calcium-activated (BK) potassium channels by somatostatin through protein dephosphorylation in rat pituitary tumor cells (White et al., Nature 351, 570-573, 1991) is blocked by drugs that interfere with arachidonic acid release by phospholipase A2 and metabolism by 5-lip-oxygenase. In contrast, higher concentrations of the same drugs had no effect on BK channel gating in cell-free patches, on the inhibition of adenylyl cyclase by somatostatin, or on the stimulation of BK channels by protein dephosphorylation through a cGMP-dependent pathway (White et al., Nature 361, 263-266, 1993). Exogenous arachidonic acid (1-20 muM) stimulated BK channel activity through protein dephosphorylation as effectively as somatostatin and was also blocked by inhibitors of lipoxygenases but not by inhibitors of phospholipase A2. These results support the hypothesis that lipoxygenase metabolites of arachidonic acid are second messengers linking pertussis toxin sensitive G-proteins to protein phosphatases regulating potassium channel activity (Armstrong and White, Trends Neurosci. 15, 403-408, 1992).

Peripheral benzodiazepine binding sites in Nb 2 node lymphoma cells: effects on prolactin-stimulated proliferation and ornithine decarboxylase activity.

[3H]Ro 5-4864 binds to Nb 2 node lymphoma cells in a specific saturable and reversible fashion. Scatchard analysis of specific binding data reveals a single, homogeneous class of whole cell binding sites with a Kd of 3.94 +/- 0.22 nM and a Bmax value of 155 +/- 11 fmol (Ro 5-4864 bound)/2 x 10(6) cells. Ro 5-4864, a reported peripheral benzodiazepine receptor agonist both inhibits (10(-6) M) and potentiates (10(-9) M) the mitogenic action of prolactin on the Nb 2 node lymphoma cells. Interestingly, PK 11195, an antagonist, potentiates (10(-9) M) the mitogenic activity of prolactin in these cells. The actions of both Ro 5-4864 and PK 11195 seem to be mediated through a common receptor type since a 10(-6) M concentration of either agent will block the others potentiating action. Furthermore, the simultaneous addition of a 10(-9) M concentration of Ro 5-4864 and PK 11195 does not further increase the effect on prolactin stimulated mitogenesis. Clonazepam, a central benzodiazepine receptor agonist has no effect on prolactin-stimulated mitogenesis in this system. These data suggest that the Nb 2 node lymphoma cells possess a peripheral-type benzodiazepine receptor. In these cells, this receptor seems to serve the function of modulating the ability of the growth factor, prolactin to initiate the mitogenic process. These studies also suggest that Ro 5-4864 is functioning as a partial agonist rather than a 'pure' agonist for the peripheral benzodiazepine receptor in this system.

Distinct sequence elements control the specificity of G protein activation by muscarinic acetylcholine receptor subtypes.

Relatively little is understood concerning the mechanisms by which subtypes of receptors, G proteins and effector enzymes interact to transduce specific signals. Through expression of normal, hybrid and deletion mutant receptors in Xenopus oocytes, we determined the G protein coupling characteristics of the functionally distinct m2 and m3 muscarinic acetylcholine receptor (mAChR) subtypes and identified the critical receptor sequences responsible for G protein specificity. Activation of a pertussis toxin insensitive G protein pathway, leading to a rapid and transient release of intracellular Ca2+ characteristic of the m3 receptor, could be specified by the transfer of as few as nine amino acids from the m3 to the m2 receptor. In a reciprocal manner, transfer of no more than 21 residues from the m2 to the m3 receptor was sufficient to specify activation of a pertussis toxin sensitive G protein coupled to a slow and oscillatory Ca2+ release pathway typical of the m2 subtype. Notably, these critical residues occur within the same region of the third cytoplasmic domain of functionally distinct mAChR subtypes.