Stimulation of B lymphocytes through their antigen receptor (BCR) results in rapid increases in tyrosine phosphorylation on a number of proteins and induces both an increase of phosphatidylinositol and mobilization of cytoplasmic free calcium. The BCR associates with two classes of tyrosine kinase: Src‐family kinase (Lyn, Fyn, Blk or Lck) and Syk kinase. To dissect the functional roles of these two types of kinase in BCR signaling, lyn‐negative and syk‐negative B cell lines were established. Syk‐deficient B cells abolished the tyrosine phosphorylation of phospholipase C‐gamma 2, resulting in the loss of both inositol 1,4,5‐trisphosphate (IP3) generation and calcium mobilization upon receptor stimulation. Crosslinking of BCR on Lyn‐deficient cells evoked a delayed and slow Ca2+ mobilization, despite the normal kinetics of IP3 turnover. These results demonstrate that Syk mediates IP3 generation, whereas Lyn regulates Ca2+ mobilization through a process independent of IP3 generation.

A group of membrane-associated guanine nucleotide binding proteins (G-proteins) are essential for transducing signals generated at cell-surface receptors into changes in cellular function and metabolism. These proteins are a complex of three subunits designated alpha, beta and gamma. The alpha-subunit is responsible for binding guanine nucleotides and seems to be characteristic of each protein. Transducin, a member of this protein family, mediates visual transduction by coupling the signal of photolysed rhodopsin with activation of a cyclic GMP phosphodiesterase. We have now cloned and sequenced the complementary DNA encoding the alpha-subunit of bovine retinal transducin and from this we have deduced the complete amino-acid sequence. The transducin alpha-subunit shares several homologous amino-acid sequences with ras gene products. The homologous segments correspond mostly to the regions thought to be involved in the guanine nucleotide binding and GTPase activity of ras proteins and to the ADP-ribosylation sites of the transducin alpha-subunit.

Interactions of G-protein ␣ (G␣) and ␤␥ subunits (G␤␥) with N-(␣ 1B ) and P/Q-type (␣ 1A ) Ca 2؉ channels were investigated using the Xenopus oocyte expression system. G i3 ␣ was found to inhibit both N-and P/Q-type channels by receptor agonists, whereas G␤ 1 ␥ 2 was responsible for prepulse facilitation of N-type channels. L-type channels (␣ 1C ) were not regulated by G␣ or G␤␥. For N-type, prepulse facilitation mediated via G␤␥ was impaired when the cytoplasmic I-II loop (loop 1) was deleted or replaced with the ␣ 1C loop 1. G␣-mediated inhibitions were also impaired by substitution of the ␣ 1C loop 1, but only when the C terminus was deleted. For P/Q-type, by contrast, deletion of the C terminus alone diminished G␣-mediated inhibition. Moreover, a chimera of L-type with the ␣ 1B loop 1 gained G␤␥-dependent facilitation, whereas an L-type chimera with the Nor P/Q-type C terminus gained G␣-mediated inhibition. These findings provide evidence that loop 1 of N-type channels is a regulatory site for G␤␥ and the C termini of P/Q-and N-types for G␣.

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1 Efonidipine, a derivative of dihydropyridine Ca 2 þ antagonist, is known to block both L-and T-type Ca 2 þ channels. It remains to be clarified, however, whether efonidipine affects other voltagedependent Ca 2 þ channel subtypes such as N-, P/Q-and R-types, and whether the optical isomers of efonidipine have different selectivities in blocking these Ca 2 þ channels, including L-and T-types. 2 To address these issues, the effects of efonidipine and its R(À)-and S( þ )-isomers on these Ca 2 þ channel subtypes were examined electrophysiologically in the expression systems using Xenopus oocytes and baby hamster kidney cells (BHK tk-ts13). 3 Efonidipine, a mixture of R(À)-and S( þ )-isomers, exerted blocking actions on L-and T-types, but no effects on N-, P/Q-and R-type Ca 2 þ channels. 4 The selective blocking actions on L-and T-type channels were reproduced by the S( þ )-efonidipine isomer. 5 By contrast, the R(À)-efonidipine isomer preferentially blocked T-type channels. 6 The blocking actions of efonidipine and its enantiomers were dependent on holding potentials. 7 These findings indicate that the R(À)-isomer of efonidipine is a specific blocker of the T-type Ca 2 þ channel.

The primary structure of the a-subunit of the adenylate cyclase-inhibiting G-protein (G,) has been deduced from the nucleotide sequence of cloned DNA complementary to the bovine cerebral mRNA encoding the polypeptide. A much higher degree of amino acid sequence homology is observed between the ix-subunits of G, and transducin (68%) than between those of G, and the adenylate cyclase-stimulating G-protein (G,) (43%) or between those of transucin and G, (42%

The alpha subunits of Gq family G proteins, GL1 alpha (G14 alpha), GL2 alpha(G11 alpha), and Gq alpha were expressed with G protein beta 1 and gamma 2 subunits in insect cells using a baculovirus system. The trimeric forms of G proteins, GL1 (GL1 alpha beta gamma), GL2 (GL2 alpha beta gamma), and Gq (Gq alpha beta gamma), were solubilized by 1% sodium cholate and purified by sequential chromatography on three kinds of columns. GL1, GL2, and Gq activated phospholipase C-beta purified from bovine brain in the presence of aluminum fluoride to the same extent. Muscarinic acetylcholine receptor m1 subtype stimulated the guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to GL1, GL2, and Gq in the presence of similar concentrations of carbamylcholine. When m1 receptor, G protein, and phospholipase C-beta were reconstituted in lipid vesicles, each subtype of Gq family G proteins mediated the activation of phospholipase C-beta by carbamylcholine in the presence of either 1 microM GTP gamma S or 1 mM GTP. Phospholipase C-beta stimulated the GTPase activity of GL1, GL2, and Gq in the presence of m1 receptor and carbamylcholine but did not stimulate the GTPase activity of GO. Protein kinase C phosphorylated m1 receptor and phospholipase C-beta, but the phosphorylation did not significantly affect the ability of the m1 receptor to stimulate phospholipase C-beta in the reconstitution system of purified proteins.