Fluorescence recovery after photobleaching of muscarinic receptors and G protein subunits tagged with cyan or yellow fluorescent protein showed that receptors and G proteins were mobile and not immobilized on the cell membrane. The cyan fluorescent protein-tagged G␣ and yellow fluorescent protein-tagged G␤ subunits were used to develop sensors that coupled selectively with the M2 and M3 muscarinic receptors. In living Chinese hamster ovary cells, imaging showed that sensors emitted a fluorescence resonance energy transfer signal that was abrogated on receptor activation. When sequentially activated with highly expressed muscarinic receptors and endogenous receptors expressed at low levels, sensor molecules were sensitive to the sequence of activation and the receptor numbers. The results distinguish between models proposing that receptor and G protein types interact freely with each other on the cell membrane or that they function as mutually exclusive multimolecular complexes by providing direct support for the former model in these cells.G proteins and their receptors have been studied extensively, but it is still not known whether they are immobilized at certain locations on the cell membrane or are capable of diffusing to various regions on the cell membrane. By tagging a muscarinic receptor, a G␣ subunit, and the G␤␥ complex with CFP 1 or YFP and examining living cells with fluorescence recovery after photobleaching, we have determined that the receptor and G protein subunits are mobile. The mobility of the signaling molecules, however, did not indicate whether particular receptors and G proteins occur in mutually exclusive multimolecular complexes or freely diffuse to collide with each other. A longstanding model proposes that specificity is achieved by mechanisms that allow certain receptor types and G protein types to be associated in exclusive multimolecular complexes (1, 2). It has been proposed that receptors and G proteins exist as complexes even in the absence of an agonist stimulus and that G protein activation occurs in isolation from other such molecular assemblies (2, 3). In a further extension of this model, it has been suggested that receptor-G protein complexes persist during signaling activity (4). These models would explain how mammalian cells respond to extracellular signals with specificity, although they often express a variety of different receptor and G protein subunit types (5). Such a mechanism would aid the rapid response to the initial signal because of the association of appropriate receptor and G protein types before activation. Evidence for the presence of assemblies of receptors and G proteins in cell membranes and their potential interaction with adaptor or scaffolding proteins have also been thought to promote such receptor-G protein molecular assemblies (4, 6 -9).An alternative model predicts that receptor types and G protein types are mobile on the cell membrane, colliding with each other freely (1, 10). For optimal determination of which of these mechanisms regulate recept...

Muscarinic acetylcholine receptors modulate the function of a variety of effectors through heterotrimeric G proteins. A prenylated peptide specific to the G protein ␥5 subunit type inhibits G protein activation by the M2 muscarinic receptor in a reconstitution assay. Scrambling the amino acid sequence of the peptide significantly reduces the efficacy of the peptide. The peptide does not disrupt the G protein heterotrimer. In cultured sympathetic neurons, the ␥5 peptide inhibits modulation of Ca 2؉ current by the M4 receptor. Peptide activity is specific, the scrambled peptide and peptides specific to two other members of the G protein ␥ subunit family are significantly less effective. The ␥5 peptide has no effect on Ca 2؉ current modulation by the ␣2-adrenergic and somatostatin receptors. In addition, the ␥5 peptide inhibits muscarinic receptor signaling in spinal cord slices with specificity. These results support a specific role for G protein ␥ subunit types in signal transduction, most likely at the receptor-G protein interface.The G protein ␥ subunits are a family of 11 proteins with varying levels of homology to each other and different patterns of expression in mammalian tissues (1). Although the G protein ␤␥ complex has been shown to directly modulate effector function and is required for receptor interaction of the G protein, the individual functions of these ␥ subunits are still unclear. Reconstitution assays with rhodopsin and Gt indicated that G protein coupling with a receptor involves specific contact of the ␥1 subunit COOH terminal with the receptor (2, 3). To test whether the COOH-terminal domains of other ␥ subunits are involved in receptor interaction we have examined the effect of a peptide from the ␥5 subunit type on muscarinic receptor signaling. ␥5 is expressed abundantly in the heart similar to the muscarinic receptor, M2 (4, 5). We examined the effect of the ␥5 COOH-terminal peptide on the activation of G i2 reconstituted with the M2 receptor. To examine the effect of the peptide in cells, we injected a peptide specific to the ␥5 COOH terminus into superior cervical ganglion (SCG) 1 neurons and measured receptor modulation of N-type Ca 2ϩ current (I ca ). SCG neurons contain the M1 and M4 muscarinic receptors which inhibit N-type Ca 2ϩ channels through G q and G o , respectively (6, 7). SCG neurons also contain ␣2-adrenergic and somatostatin receptors that inhibit I ca through G o (6). This variety of receptors modulating the activity of a common effector allowed us to assess the specificity of the ␥5 peptide action. The effect of ␥5 peptides as well as peptides from ␥7 and ␥12, on these pathways was examined. Finally, to test the effect of the ␥5 peptide on the central nervous system, we introduced the ␥5 peptide into postsynaptic neurons in a spinal cord slice and measured the modulation of glutamate receptor mediated synaptic current by muscarinic and ␣2-adrenergic receptors (8). MATERIALS AND METHODS Cells and Reagents-[3 H]N-methylscopolamine and [ 35 S]GTP␥S were from NEN Life Scienc...

The effects of transgenic overexpression of glycogen synthase in different types of fast-twitch muscle fibers were investigated in individual fibers from the anterior tibialis muscle. Glycogen synthase was severalfold higher in all transgenic fibers, although the extent of overexpression was twofold greater in type IIB fibers. Effects of the transgene on increasing glycogen and phosphorylase and on decreasing UDP-glucose were also more pronounced in type IIB fibers. However, in any grouping of fibers having equivalent malate dehydrogenase activity (an index of oxidative potential), glycogen was higher in the transgenic fibers. Thus increasing synthase is sufficient to enhance glycogen accumulation in all types of fast-twitch fibers. Effects on glucose transport and glycogen synthesis were investigated in experiments in which diaphragm, extensor digitorum longus (EDL), and soleus muscles were incubated in vitro. Transport was not increased by the transgene in any of the muscles. The transgene increased basal [(14)C]glucose into glycogen by 2.5-fold in the EDL, which is composed primarily of IIB fibers. The transgene also enhanced insulin-stimulated glycogen synthesis in the diaphragm and soleus muscles, which are composed of oxidative fiber types. We conclude that increasing glycogen synthase activity increases the rate of glycogen synthesis in both oxidative and glycolytic fibers, implying that the control of glycogen accumulation by insulin in skeletal muscle is distributed between the glucose transport and glycogen synthase steps.

No abstract