Association of particulate transglutaminase with the nerve-muscle synapse.

removed. air dried. incubated in 7 X 1 0 I ' M-l"l-~abclkd cyanopindolol (CY P). washed, rapidly dried and subjected to autoradiography t o detect the presence of /3-adrenergic receptors [ 8 I. Autoradiography of polyester filter replicas after fusion ot COS cells with spheroplasts containing pPAR revealed punctate areas with high density "'I-CYP binding, consistent with induction o f /3-adrcnergic receptor expression from pPAR [2]. T-antigen was essential to the production of the high-density binding areas since CV-1 cells (the progenitor cell line for COS) did not produce any of these areas. We found that the COS-M6 line produced several times the number o f high-density binding sites than COS-7 cell5 purchased from ATCC. Levels of T-antigen vary for COS cell lines and in individual cells [S]. It is thus conceivable that differences in T-antigen levels in COS cells could limit both the proportion of cells expressing P-adrenergic receptors and the level of that expression after spheroplast fusion. If a COS cell receives more than one cDNA for which expression is dependent upon T-antigen. it is possible that the occurrence and level o f cDNA expression depends on competition for a limited pool o f T-antigen. When spheroplasts containing p P A R were diluted with spheroplasts containing pSVL, the number of punctate binding sites found on filter autoradiographs was proportional to the proportion o f spheroplasts containing pPAR 121. If expression o f high-density '?'I-CY P binding areas was limited by cornpetition for T-antigen in COS cells we would anticipate that mixtures of p/3AR-spheroplasts with pUC18 instead of pSVL would produce an increased number o f high-density binding sites. Howcvcr, we found that the relationship between the number of high-density binding areas and the proportion of p P A R in the spheroplast mixture was identical ( I .6 spots/filter per "A, p P A R spheroplasts). whether pUC-18 or pSVL spheroplasts were used. This result suggests that competition for T-antigen levels in individual COS cells does not limit receptor expression from pPAR. Thus low abundance cDNAs should be able to be expressed without changing conditions to reduce T-antigen competition, e.g. by diluting with 'carrier' spheroplasts. Ongoing experiments are addressing related issues such as whether COS cells can express more than one plasmid after spheroplast fusion.