Activation of transfected M1 or M3 muscarinic acetylcholine receptors induces cell-cell adhesion of Chinese hamster ovary cells expressing endogenous cadherins.
Expression of endogenous cadherins by Chinese hamster ovary (CHO) cells has not been previously reported. However, we observed that CHO cells adhere to one another upon activation of transfected muscarinic acetylcholine receptors (mAChR), suggesting that the cells express endogenous cadherins. A 160-base pair RT-PCR product with 100% homology to the cytoplasmic domain of human E-cadherin was amplified from CHO cells. A second RT-PCR product amplified from these cells has 92% homology to the cytoplasmic domain of human cadherin-9 and 86% homology to the cytoplasmic domain of human cadherin-6. Western blotting indicates that CHO cells express a 165-kDa protein recognized by E-cadherin antibodies and a 120-kDa protein recognized by an antibody to the cadherin C-terminus sequence. The ability of transfected mAChR subtypes to regulate cadherin-mediated adhesion of CHO cells was tested by measuring the permeation of horseradish peroxidase across confluent CHO cell monolayers, by microscopic examination of the cells, and by aggregation assays. Cell-cell adhesion is induced within 15 min of activating transfected M1 or M3 mAChR which functionally couple to protein kinase C (PKC). In contrast, CHO cell adhesion is not affected by activating transfected M2 mAChR which functionally couple to other effectors. Activation of PKC with phorbol esters also induces cell-cell adhesion of all CHO sublines tested. Immunofluorescence assays reveal that endogenous cadherins redistribute on the plasma membrane of CHO cells following mAChR or PKC activation. Inactivation of cadherins by removal of extracellular Ca2+ abrogates adhesion induced by mAChR or PKC activation. Our demonstration that activation of only odd-numbered mAChR subtypes induces cadherin-mediated adhesion suggests that the unique responses of cells to M1 or M3 mAChR stimulation may involve cadherin activation.
Shafer
S H
SH
Laboratory of Neurobiology, Guthrie Research Institute, Sayre, Pennsylvania, 18840, USA.
Puhl
H L
HL
Phelps
S H
SH
Williams
C L
CL
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D015820:Cadherins / Q000096:biosynthesis* / Q000235:genetics; D002448:Cell Adhesion; D002460:Cell Line; D006224:Cricetinae; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D043643:Receptor, Muscarinic M1; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000378:metabolism*
D015820:Cadherins; D043643:Receptor, Muscarinic M1; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D011493:Protein Kinase C