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Gap junction protein homologue from Arabidopsis thaliana: evidence for connexins in plants.

ABSTRACT: An Arabidopsis thaliana library constructed in the pBluescript expression vector lambda ZAP II (Stratagene) was screened with three affinity-purified antibodies raised against (i) rat liver connexin 32, (ii) a polypeptide from soybean root cells that migrates with a molecular mass of 29 kDa in SDS/polyacrylamide gels and is immunologically related to rat liver connexin, and (iii) a synthetic peptide corresponding to a sequence in rat liver connexin 32. A single clone was obtained whose gene product demonstrated immunological crossreactivity with all three reagents. The cDNA from this clone contained 1171 base pairs and coded for a protein of 280 amino acids with a calculated molecular mass of 32,339 Da (migrates as a 29-kDa polypeptide in SDS/polyacrylamide gels). The sequence homology observed between the 32-kDa polypeptide of Arabidopsis and connexin 32 from rat liver, in conjunction with observed similarities in predicted number and distribution of hydrophobic domains, sites for posttranslational modification, and basic pI, provides strong evidence that the biological range for connexin-type proteins may now be considered to include the plant kingdom.

SUBMITTER: Meiners S

PROVIDER: S-EPMC51609 | biostudies-other | 1991 May

REPOSITORIES: biostudies-other

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Gap junction protein homologue from Arabidopsis thaliana: evidence for connexins in plants.

Meiners S S Xu A A Schindler M M

Proceedings of the National Academy of Sciences of the United States of America 19910501 10

An Arabidopsis thaliana library constructed in the pBluescript expression vector lambda ZAP II (Stratagene) was screened with three affinity-purified antibodies raised against (i) rat liver connexin 32, (ii) a polypeptide from soybean root cells that migrates with a molecular mass of 29 kDa in SDS/polyacrylamide gels and is immunologically related to rat liver connexin, and (iii) a synthetic peptide corresponding to a sequence in rat liver connexin 32. A single clone was obtained whose gene prod ...[more]

PMID: 1851993

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Project description:Ca2+/calmodulin (Ca2+/CaM) interaction with connexins (Cx) is well-established; however, the mechanistic basis of regulation of gap junction function by Ca2+/CaM is not fully understood. Ca2+/CaM is predicted to bind to a domain in the C-terminal portion of the intracellular loop (CL2) in the vast majority of Cx isoforms and for a number of Cx-s this prediction has proved correct. In this study, we investigate and characterise both Ca2+/CaM and apo-CaM binding to selected representatives of each of the α, β and γ connexin family to develop a better mechanistic understanding of CaM effects on gap junction function. The affinity and kinetics Ca2+/CaM and apo-CaM interactions of CL2 peptides of β-Cx32, γ-Cx35, α-Cx43, α-Cx45 and α-Cx57 were investigated. All five Cx CL2 peptides were found to have high affinity for Ca2+/CaM with dissociation constants (Kd(+Ca)) from 20 to 150 nM. The limiting rate of binding and the rates of dissociation covered a broad range. In addition, we obtained evidence for high affinity Ca2+-independent interaction of all five peptides with CaM, consistent with CaM remaining anchored to gap junctions in resting cells. However, for the α-Cx45 and α-Cx57 CL2 peptides, Ca2+-dependent association at resting [Ca2+] of 50-100 nM is indicated in these complexes as one of the CaM Ca2+ binding sites displays high affinity with Kd of 70 and 30 nM for Ca2+, respectively. Furthermore, complex conformational changes were observed in peptide-apo-CaM complexes with the structure of CaM compacted or stretched by the peptide in a concentration dependent manner suggesting that the CL2 domain may undergo helix-to-coil transition and/or forms bundles, which may be relevant in the hexameric gap junction. We demonstrate inhibition of gap junction permeability by Ca2+/CaM in a dose dependent manner, further cementing Ca2+/CaM as a regulator of gap junction function. The motion of a stretched CaM-CL2 complex compacting upon Ca2+ binding may bring about the Ca2+/CaM block of the gap junction pore by a push and pull action on the CL2 C-terminal hydrophobic residues of transmembrane domain 3 (TM3) in and out of the membrane.

Project description:BACKGROUND AND AIMS:Connexins and their cell membrane channels contribute to the control of cell proliferation and compartmental functions in breast glands and their deregulation is linked to breast carcinogenesis. Our aim was to correlate connexin expression with tumor progression and prognosis in primary breast cancers. MATERIALS AND METHODS:Meta-analysis of connexin isotype expression data of 1809 and 1899 breast cancers from the Affymetrix and Illumina array platforms, respectively, was performed. Expressed connexins were also monitored at the protein level in tissue microarrays of 127 patients equally representing all tumor grades, using immunofluorescence and multilayer, multichannel digital microscopy. Prognostic correlations were plotted in Kaplan-Meier curves and tested using the log-rank test and cox-regression analysis in univariate and multivariate models. RESULTS:The expression of GJA1/Cx43, GJA3/Cx46 and GJB2/Cx26 and, for the first time, GJA6/Cx30 and GJB1/Cx32 was revealed both in normal human mammary glands and breast carcinomas. Within their subfamilies these connexins can form homo- and heterocellular epithelial channels. In cancer, the array datasets cross-validated each other's prognostic results. In line with the significant correlations found at mRNA level, elevated Cx43 protein levels were linked with significantly improved breast cancer outcome, offering Cx43 protein detection as an independent prognostic marker stronger than vascular invasion or necrosis. As a contrary, elevated Cx30 mRNA and protein levels were associated with a reduced disease outcome offering Cx30 protein detection as an independent prognostic marker outperforming mitotic index and necrosis. Elevated versus low Cx43 protein levels allowed the stratification of grade 2 tumors into good and poor relapse free survival subgroups, respectively. Also, elevated versus low Cx30 levels stratified grade 3 patients into poor and good overall survival subgroups, respectively. CONCLUSION:Differential expression of Cx43 and Cx30 may serve as potential positive and negative prognostic markers, respectively, for a clinically relevant stratification of breast cancers.

Dataset Information

Gap junction protein homologue from Arabidopsis thaliana: evidence for connexins in plants.

Publications

Gap junction protein homologue from Arabidopsis thaliana: evidence for connexins in plants.

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