Identification of a basolateral sorting signal for the M3 muscarinic acetylcholine receptor in Madin-Darby canine kidney cells.

Muscarinic acetylcholine receptors (mAChRs) can be differentially localized in polarized cells. To identify potential sorting signals that mediate mAChR targeting, we examined the sorting of mAChRs in Madin-Darby canine kidney cells, a widely used model system. Expression of FLAG-tagged mAChRs in polarized Madin-Darby canine kidney cells demonstrated that the M(2) subtype is sorted apically, whereas M(3) is targeted basolaterally. Expression of M(2)/M(3) receptor chimeras revealed that a 21-residue sequence, Ser(271)-Ser(291), from the M(3) third intracellular loop contains a basolateral sorting signal. Substitution of sequences containing the M(3) sorting signal into the homologous regions of M(2) was sufficient to confer basolateral localization to this apical receptor. Sequences containing the M(3) sorting signal also conferred basolateral targeting to M(2) when added to either the third intracellular loop or the C-terminal cytoplasmic tail. Furthermore, addition of a sequence containing the M(3) basolateral sorting signal to the cytoplasmic tail of the interleukin-2 receptor alpha-chain caused significant basolateral targeting of this heterologous apical protein. The results indicate that the M(3) basolateral sorting signal is dominant over apical signals in M(2) and acts in a position-independent manner. The M(3) sorting signal represents a novel basolateral targeting motif for G protein-coupled receptors.

Asymmetric distribution of muscarinic acetylcholine receptors in Madin-Darby canine kidney cells.

We have characterized the muscarinic ACh receptors (mAChRs) expressed in Madin- Darby canine kidney (MDCK) strain II epithelial cells. Binding studies with the membrane-impermeable antagonist N-[(3)H]methylscopolamine demonstrated that mAChRs are approximately 2.5 times more abundant on the basolateral than on the apical surface. Apical, but not basolateral, mAChRs inhibited forskolin-stimulated adenylyl cyclase activity in response to the agonist carbachol. Neither apical nor basolateral mAChRs exhibited detectable carbachol-stimulated phospholipase C activity. Carbachol application to the apical or the basolateral membrane resulted in a threefold increase in intracellular Ca(2+) concentration, which was completely inhibited by pertussis toxin on the apical side and partially inhibited on the basolateral side. RT-PCR analysis showed that MDCK cells express the M(4) and M(5) receptor mRNAs. These data suggest that M(4) receptors reside on the apical and basolateral membranes of polarized MDCK strain II cells and that the M(5) receptor may reside in the basolateral membrane of a subset of cells.

Molecular analysis of the regulation of muscarinic receptor expression and function.

We have investigated the molecular mechanisms involved in the regulation of muscarinic acetylcholine receptor gene expression and localization and generated knockout mice to study the role of the M1 muscarinic receptor in vivo. We have used the MDCK cell system to demonstrate that different subtypes of mAChR can be targeted to different regions of polarized cells. We have also examined the developmental regulation of mAChR expression in the chick retina. Early in development, the M4 receptor is the predominant mAChR while the levels of the M2 and M3 receptors increase later in development. The level of M2 receptor is also initially very low in retinal cultures and undergoes a dramatic increase over several days in vitro. The level of M2 receptor can be increased by a potentially novel, developmentally regulated, secreted factor produced by retinal cells. The promoter for the chick M2 receptor gene has been isolated and shown to contain a site for GATA-family transcription factors which is required for high level cardiac expression. The M2 promoter also contains sites which mediate induction of transcription in neural cells by neurally active cytokines. We have generated knockout mice lacking the M1 receptor and shown that these mice do not exhibit pilocarpine-induced seizures and muscarinic agonist-induced suppression of the M-current potassium channel in sympathetic neurons.

GABAA receptor subunit expression and assembly in cultured rat cerebellar granule neurons.

The assembly of multisubunit GABAA receptors in specific neuronal populations is a complex process which is poorly understood. To begin to examine receptor assembly, alpha 1, beta 2/3, and gamma 2 subunit polypeptide expression and association, as well as receptor binding, were examined in cultured rat cerebellar granule neurons. Western blots revealed two alpha 1-immunoreactive proteins. A 39 kDa species was maximal at 2 days in culture and subsequently declined. In contrast, a 51 kDa polypeptide, the anticipated size of the mature alpha 1 subunit, was first detected at 4 days and increased throughout the culture period. Additional studies demonstrated that the beta 2/3 and gamma 2 subunits were detectable at 2 days and attained maximal levels by 6 days. The level of [3H]Ro15-1788 binding, a measure of assembled receptors, rose in parallel with the increases in the 51 kDa alpha 1, beta 2/3 and gamma 2 subunits. Moreover, the 51 kDa alpha 1, beta 2/3, and gamma 2 subunits were associated in receptor complexes. However, immunohistochemical studies demonstrated the presence of substantial intracellular subunit staining. This finding suggest that only some of the subunits expressed in granule neurons contribute to functional GABAA receptors on the cell surface.

GABAA receptor subunit polypeptides increase in parallel but exhibit distinct distributions in the developing rat cerebellum.

The GABAA receptor, a multisubunit ligand-gated ion channel, plays a central role in cell-cell communication in the developing and adult nervous system. Although the developmental expression of mRNAs encoding many subunit isoforms has been extensively characterized throughout the central nervous system, little is known concerning the relationship between subunit mRNA and polypeptide expression. To address this issue, we examined the developmental expression of the alpha 1, beta 2/3, and gamma 2 subunit polypeptides, subunits that are thought to coassemble in many brain regions. Western blot analysis using subunit-specific antibodies revealed that the levels of these polypeptides in both the cerebral cortex and cerebellum increased severalfold during the second postnatal week. Whereas polypeptide expression in the cerebellum paralleled that of the corresponding subunit mRNAs, increases in beta 2/3 and gamma 2 polypeptide expression in the cerebral cortex occurred in the absence of detectable changes in the mRNA levels. To determine whether the increases in subunit polypeptide expression in the cerebellum were accompanied by changes in distribution, immunohistochemistry was performed. These studies demonstrated that the subunits exhibited different but partially overlapping distributions that remained constant throughout postnatal development. Our findings suggest that although GABAA receptor subunit polypeptide expression may be regulated primarily at the level of the mRNA, additional regulatory mechanisms may play a role. Furthermore, the observation that subunit distribution remains constant in the cell bodies of cerebellar Purkinje neurons, which express the alpha 1, beta 2, beta 3, and gamma 2 subunit mRNAs exclusively, suggests that GABAA receptor subunit composition in this cell population does not change during postnatal maturation.