A BSTR ACTA recently cloned isoform of cGMPdependent protein kinase (cGK), designated type II, was implicated as the mediator of cGMP-provoked intestinal Cl ؊ secretion based on its localization in the apical membrane of enterocytes and on its capacity to activate cystic fibrosis transmembrane conductance regulator (CFTR) Cl ؊ channels. In contrast, the soluble type I cGK was unable to activate CFTR in intact cells, although both cGK I and cGK II could phosphorylate CFTR in vitro. To investigate the molecular basis for the cGK II isotype specificity of CFTR channel gating, we expressed cGK II or cGK I mutants possessing different membrane binding properties by using adenoviral vectors in a CFTR-transfected intestinal cell line, and we examined the ability of cGMP to phosphorylate and activate the Cl ؊ channel. Mutation of the cGK II N-terminal myristoylation site (Gly 2 3 Ala) reduced cGK II membrane binding and severely impaired cGK II activation of CFTR. Conversely, a chimeric protein, in which the N-terminal membraneanchoring domain of cGK II was fused to the N terminus of cGK I␤, acquired the ability to associate with the membrane and activate the CFTR Cl ؊ channel. The potency order of cGK constructs for activation of CFTR (cGK II > membranebound cGK I chimer > > nonmyristoylated cGK II > cGK I␤) correlated with the extent of 32 P incorporation into CFTR observed in parallel measurements. These results strongly support the concept that membrane targeting of cGK is a major determinant of CFTR Cl ؊ channel activation in intact cells.

The development of effective receptor-targeted nonviral vectors for use in vivo is complicated by a number of technical problems. One of these is the low efficiency of the conjugation procedures used to couple protein ligands to the DNA condensing carrier molecules. We have made and characterized a multi-domain protein (SPKR) 4 inv, that is designed to target plasmid DNA to ␤ 1 integrins in remodeling tissue. It contains a nonspecific DNA-binding domain (SPKR) 4 , a rigid ␣-helical linker, and the C-terminal ␤ 1 integrin binding domain (aa 793-987) of the Yersinia pseudotuberculosis invasin protein. (SPKR) 4 inv could be purified at high yields using a bacterial expression system. We show that (SPKR) 4 inv binds with high affinity to both plasmid DNA and ␤ 1 integrins. In a cell attachment assay, the apparent affinity of (SPKR) 4 inv for ␤ 1 integrins is three orders of magnitude