Guanine nucleotide binding (G) proteins play pivotal roles in the cellular physiology of ion channel regulation and signal transduction coupled to hormones and their receptors. G proteins are heterotrimers composed of individual alpha, beta, and gamma subunits. A super gene family encodes at least sixteen distinct a subunits for transducing G proteins which are structurally similar. Different G proteins are often found in the same cell. However, the cellular mechanisms that allow for 1) the expression of different G proteins and 2) the specificity required for different G proteins to couple their effectors in diverse tissues is not understood. To understand these mechanisms we have utilized the porcine LLC-PK1 and A6 amphibian kidney cells as models to study G protein coupled processes in epithelia. We have found two geographically defined activities in polar LLC-PK1 cells: 1) at the basolateral membrane, vasopressin binding its V2 receptor results in the activation of adenylyl cyclase and the generation of cAMP; and 2) at the apical membrane, the transport of Na+ is mediated by an amiloride-sensitive Na+ channel. Both of these activities are developmentally expressed in LLC-PK1L cells and are regulated by pertussis toxin-sensitive processes which implicated the involvement of a subset of G proteins (Gi) in these events. Three described Gi proteins (Gi-1, Gi-2, and, Gi-3) are distinguished by a unique alpha(i) subunit. Only Gi-2 and Gi-3 are found in LLC-PK1 cells. We propose to examine the mechanism of G protein alpha(i) subunit gene expression, targeting, and coupling to effectors in these kidney cells. The rationale for such studies is based on our recent findings that polar LLC-PK1 cells: 1) undergo a developmental expression of distinct alpha(i)-2 and alpha(i)-3 subunit gene products during culture and 2) these subunits are differentially expressed and targeted to basolateral (alpha(i)-2) and apical (alpha(i)-3) membrane sites where they interact with and modulate their respective enzyme and ion channel effectors. In A6 cells activated alpha(i)-3 but not alpha(i)-2 activated apical Na+ channels in excised patches. Understanding the factors which control Gi protein expression, targeting, and effector coupling is an important and necessary step in elucidating the complex physiology of transepithelial transport processes in renal epithelial cells. For targeting and effector coupling studies, we have established methods to express or produce chimeric alpha(i) subunits, localize, and monitor their effector interactions. For gene expression studies, we have isolated both the porcine alpha(i)-2 and alpha(i)-3 genes and fully characterized their promoters. These genes are likely to be under the control of hormones and growth factors which are known or suspected to alter ion transport in renal epithelia. We have demonstrated that the alpha(i)-3 gene is transcriptionally regulated by aldosterone. Determining which discrete DNA sequences are requisite for alpha(i)-2 and alpha(i)-3 gene regulation hormones and growth factors will provide the means to identify, isolate, and clone the nuclear transcription factor (NTF) proteins which activate them. The information gained from these studies should provide fundamental knowledge concerning the mechanisms of Gi protein expression and effector coupling which influence kidney cellular physiology in health and disease.
