The mechanismS whereby cyclic nucleotide phosphodiesterases (PDEs) are targeted to particular membrane sites or to the cytosol, and the functional significance of the specific intracellular location of PDE isoforms are not understood. cDNAs encoding adipocyte-type and myocardial-type cGMP-inhibited PDE isoforms (cGIP1 and cGIP2, respctively) have recently been cloned. The adipocyte cGI-PDE is particulate, whilst the myocardial isoform is found in the cytosol and in association with the sarcoplasmic reticulum. The N-termini of these cGI-PDEs contain hydrophobic putative membrane association domains and several consensus protein kinase A phosphorylation sites, which may play a role in regulation of the enzyme. To investigate the function of this domain, full length human cardiac cGI PDE (HcGIP2) and a series of N-terminal truncated HcGIP2 forms will be expressed, transiently in the SV40 transformed monkey kidney cell line, COS-7, and stably in an insulin-responsive preadipocyte cell line, 3T3-L1. This will enable us to define the minimum sequence necessary for membrane association, to determine the intracellular location/distribution of the enzyme and also to determine whether alteration in the cellular location of the PDE affects regulation of the PDE, or the processes it regulates. Similar studies will also be conducted with a recently cloned mouse adipocyte cGI-PDE (McGIP1). Preliminary to these studies it was necessary to introduce a Flag epitope tag into the protein sequence of HcGIP2 to enable selective recognition of the PDE following transfection into COS-7 and 3T3-L1 cell lines.
