Localization of muscarinic cholinergic receptor subtypes in human epidermis and keratinocyte cell culture

Ndoye, Assane; Buchli, Rico; Greenberg, Brandon M.; Nguyen, Vu Thuong; Lawry, Monica; Webber, Robert J.; Grando, Sergei A.

doi:10.1016/s0923-1811(98)83637-2

Cited by 3 publications

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“…Redistribution of M 1 immunoreactivity to the leading edge of KCs preceded crescent shape formation required for directional migration, indicating that among the M 1 -M 5 mAChR subtypes expressed in human KCs (45,54), the M 1 was primarily involved. Both coupling of M 1 to the Ras/Raf/MAP kinase pathway and coupling of this pathway to up-regulation of integrin ␣ 2 have been demonstrated in other cell types (30,55).…”

Section: Discussionmentioning

confidence: 99%

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The Ras/Raf-1/MEK1/ERK Signaling Pathway Coupled to Integrin Expression Mediates Cholinergic Regulation of Keratinocyte Directional Migration

Chernyavsky

Arredondo

Karlsson

et al. 2005

Journal of Biological Chemistry

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The physiologic mechanisms that determine directionality of lateral migration are a subject of intense research. Galvanotropism in a direct current (DC) electric field represents a natural model of cell re-orientation toward the direction of future migration. Keratinocyte migration is regulated through both the nicotinic and muscarinic classes of acetylcholine (ACh) receptors. We sought to identify the signaling pathway mediating the cholinergic regulation of chemotaxis and galvanotropism. The pharmacologic and molecular modifiers of the Ras/Raf-1/MEK1/ERK signaling pathway altered both chemotaxis toward choline and galvanotropism toward the cathode in a similar way, indicating that the same signaling steps were involved. The galvanotropism was abrogated due to inhibition of ACh production by hemicholinium-3 and restored by exogenously added carbachol. The concentration gradients of ACh and choline toward the cathode in a DC field were established by highperformance liquid chromatographic measurements. This suggested that keratinocyte galvanotaxis is, in effect, chemotaxis toward the concentration gradient of ACh, which it creates in a DC field due to its highly positive charge. A time-course immunofluorescence study of the membrane redistribution of ACh receptors in keratinocytes exposed to a DC field revealed rapid relocation to and clustering at the leading edge of ␣7 nicotinic and M 1 muscarinic receptors. Their inactivation with selective antagonists or small interfering RNAs inhibited galvanotropism, which could be prevented by transfecting the cells with constitutively active MEK1. The end-point effect of the cooperative signaling downstream from ␣7 and M 1 through the MEK1/ERK was an up-regulated expression of ␣ 2 and ␣ 3 integrins, as judged from the results of real-time PCR and quantitative immunoblotting. Thus, ␣7 works together with M 1 to orient a keratinocyte toward direction of its future migration. Both ␣7 and M 1 apparently engage the Ras/Raf/MEK/ERK pathway to up-regulate expression of the "sedentary" integrins required for stabilization of the lamellipodium at the keratinocyte leading edge. Human epidermal keratinocytes (KCs)2 can synthesize and secrete acetylcholine (ACh) and use it as a local hormone for an autocrine and paracrine control of their vital functions, including motility (1). ACh and its congeners are chemotactic for KCs, neurons, and other cell types (2-8). To characterize the physiologic control of keratinocyte migration, we developed an in vitro model of skin epithelialization, termed the agarose gel keratinocyte outgrowth system (AGKOS) (9). Using AGKOS, we have demonstrated that activation of the keratinocyte nicotinic ACh receptors (nAChRs) comprised by ␣7 subunits exhibit reciprocal effects on cell motility by stimulating directional (chemotaxis) and inhibiting random (chemokinesis) migration and that ACh-gated ion channels containing ␣3 subunit stimulate chemokinesis of KCs (8).More recently, ␣7 nAChR has been shown to mediate chemotaxis of vascular smooth muscle cells to...

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Section: Discussionmentioning

confidence: 99%

“…The fluorescence was examined with an Axiovert 135 fluorescence microscope (Carl Zeiss Inc., Thornwood, NY). The specificity of antibody binding was demonstrated by omitting the primary antibody or by replacing it with an irrelevant antibody of the same isotype and species as the primary antibody, as detailed elsewhere (45).…”

Section: Methodsmentioning

confidence: 99%

The Ras/Raf-1/MEK1/ERK Signaling Pathway Coupled to Integrin Expression Mediates Cholinergic Regulation of Keratinocyte Directional Migration

Chernyavsky

Arredondo

Karlsson

et al. 2005

Journal of Biological Chemistry

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“…The relative amounts of keratinocyte membrane proteins targeted by PV IgG in murine skin and adhesion molecules expressed by human KC in vitro were estimated by the semi-quantitative IF assay described previously (62,63). The assay is based on calculating the intensity of fluorescence pixel by pixel by dividing the summation of pixels of fluorescence intensity by the segment occupied by the pixels (i.e.…”

Section: Pemphigus and Control Igg Fractions-mentioning

confidence: 99%

Pemphigus Vulgaris IgG and Methylprednisolone Exhibit Reciprocal Effects on Keratinocytes

Nguyen

Arredondo

Chernyavsky

et al. 2004

Journal of Biological Chemistry

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“…The human M 3 muscarinic receptor was expected to migrate at a mass of 67-75 kDa based on their primary sequence and on previous reports (Figure 5.1. 6) 430 . The N540A mutant is unable to activate PLC, PLD and MAPK pathway.…”

Section: N540d and F551a Mutants Are Expressed On The Cell Surface An...mentioning

confidence: 99%

Human M3 muscarinic acetylcholine receptor protein-protein interactions: roles in receptor signaling and regualation

Borroto Escuela

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Muscarinic acetylcholine receptors (mAChRs) have been shown to mediate various functions in the central and peripheral nervous systems. These include modulation of exocrine glandular secretion, vasodilatation and smooth muscle contraction, cell proliferation or survival, neural development and synaptic plasticity. mAChRs are activated by both endogenously produced acetylcholine and exogenously administered muscarinic compounds. Pharmacological, anatomical and molecular studies have demonstrated the existence of five muscarinic receptor subtypes, denoted as muscarinic M1, M2, M3, M4 and M5, which belong to class I family of heptahelical, transmembrane G-protein coupled receptors (GPCRs). Each receptor subtypes are characterized by a distinct selectivity for heterotrimeric G protein coupling. Thus, M1, M3 and M5 are coupled to Gq/11 proteins and stimulate phospholipase C activity, resulting in the generation of the second messengers inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG), the mobilization of intracellular Ca2+ and the activation of protein kinase C (PKC). On the other hand, M2 and M4 are coupled to Gi/0 proteins, which results in the inhibition of adenilate cyclase, as well as prolonging potassium channel, non-selective cation channel, and transient receptor potential channel opening. By means of this differential set of G protein partners, mAChRs can initiate distinct signalling pathways within a same cell in order to trigger diverse, even opposed, functional outcomes in response to the same stimuli. It has been proven as well that mAChRs regulate a baste network of signalling intermediates, including small GTPase Rho, phospholipase D, phosphoinositide-3 kinase, non-receptor kinases and mitogenactivated protein kinases. Although the first proteins found to have functional interactions with mAChRs were, of course, G proteins, an increasing amount of evidence in the field suggests that this simplistic model defined as “one receptor -one G protein -one effector no longer exists. A great number of proteins have been identified as interacting with mAChRs, including GPCRs, kinases, and scaffolding proteins such as arrestin. Determining in part the signalling efficiency/specificity for mAChRs. Thus, receptors are now considered as complex signalling units, or signalosomes, that dynamically couple to multiple G proteins or other molecular entities or scaffold proteins in a temporally and spatially regulated manner, and even can form homodimers or heterodimers with distinct GPCRs or other non-GPCR membrane receptors, resulting in pharmacologically and functionally distinct receptor populations. In this work “Human M3 muscarinic acetylcholine receptor protein-protein interactions: roles in receptor signalling and regulation”, it is discuss novel mAChRs interacting partners that link the receptors to alternative signalling pathways beyond G proteins. Emphases on explaining how mAChRs regulate signal transduction pathways mediated by these proteins, including receptor dimerization have been putting out. It has been demonstrated by different approach (from resonance energy transfer to tandem affinity purification and mass spectrometry) the active role of interacting protein in mAChRs regulation and signalling. We shown that a particular complex is not necessarily of invariable composition, nor are all its building blocks uniquely associated with that specific complex. One complex may be the result not only of physical interaction between the receptor and the partners’ protein, but also of the participation of many non-“direct” associations resulting in the formation of a network that interconnects the receptor with a number of other pathways, determining receptor specificities. This allows us to address some fundamental questions concerning the importance of molecular mechanisms hidden behind the pharmacology properties for each receptor subtype.

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Localization of muscarinic cholinergic receptor subtypes in human epidermis and keratinocyte cell culture

Cited by 3 publications

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The Ras/Raf-1/MEK1/ERK Signaling Pathway Coupled to Integrin Expression Mediates Cholinergic Regulation of Keratinocyte Directional Migration

The Ras/Raf-1/MEK1/ERK Signaling Pathway Coupled to Integrin Expression Mediates Cholinergic Regulation of Keratinocyte Directional Migration

Pemphigus Vulgaris IgG and Methylprednisolone Exhibit Reciprocal Effects on Keratinocytes

Human M3 muscarinic acetylcholine receptor protein-protein interactions: roles in receptor signaling and regualation

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