Elizabeth A. Jewell-Motz

G Protein-coupled Receptor Kinase Specificity for Phosphorylation and Desensitization of α2-Adrenergic Receptor Subtypes

The α2-adrenergic receptor (α2AR) subtype α2C10 undergoes rapid agonist-promoted desensitization which is due to phosphorylation of the receptor. One kinase that has been shown to phosphorylate α2C10 in an agonist-dependent manner is the βAR kinase (βARK), a member of the family of G protein-coupled receptor kinases (GRKs). In contrast, the α2C4 subtype has not been observed to undergo agonist-promoted desensitization or phosphorylation by βARK. However, the substrate specificities of the GRKs for phosphorylating α2AR subtypes are not known. We considered that differential capacities of various GRKs to phosphorylate α2C10 and α2C4 might be a key factor in dictating in a given cell the presence or extent of agonist-promoted desensitization of these receptors. COS-7 cells were co-transfected with α2C10 or α2C4 without or with the following GRKs: βARK, βARK2, GRK5, or GRK6. Intact cell phosphorylation studies were carried out by labeling cells with 32Pi, exposing some to agonist, and purifying the α2AR by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. βARK and βARK2 were both found to phosphorylate α2C10 to equal extents (>2-fold over that of the endogenous kinases). On the other hand, GRK5 and GRK6 did not phosphorylate α2C10. In contrast to the findings with α2C10, α2C4 was not phosphorylated by any of these kinases. Functional studies carried out in transfected HEK293 cells expressing α2C10 or α2C4 and selected GRKs were consistent with these phosphorylation results. With the marked expression of these receptors, no agonist-promoted desensitization was observed in the absence of GRK co-expression. However, desensitization was imparted to α2C10 by co-expression of βARK but not GRK6, while α2C4 failed to desensitize with co-expression of βARK. These results indicate that short term agonist-promoted desensitization of α2ARs by phosphorylation is dependent on both the receptor subtype and the expressed GRK isoform.

Mutational analysis of Glu-327 of Na+-K+-ATPase reveals stimulation of86Rb+uptake by external K+

A competition assay of 86Rb+ uptake in HeLa cells transfected with ouabain-resistant Na(+)-K(+)-ATPase mutants revealed a stimulation of 86Rb+ uptake at low external concentrations (1 mM) of competitor (K+). Of the models that were tested, those that require that two K+ be bound before transport occurs gave the worst fits. Random and ordered binding schemes described the data equally well. General models in which both binding and transport were allowed to be cooperative yielded parameter errors larger than the parameters themselves and could not be utilized. Models that assumed noncooperative transport always showed positive cooperativity in binding. E327Q and E327L mutated forms of rat alpha 2 had lower apparent affinities for the first K+ bound than did wild-type rat alpha 2 modified to be ouabain resistant. The mutations did not affect the apparent affinity of the second K+ bound. Models that assumed noncooperativity in binding always showed positively cooperative transport, i.e., enzymes with two K+ bound had a higher flux than those with one K+ bound. Increases in external Na+ decreased the apparent affinity for K+ for all models and decreased the ratio of the apparent influx rate constants for E327L.

Na,K-ATPase: Cardiac Glycoside Binding and Functional Importance of Negatively Charged Amino Acids of Transmembrane Regions

Na+/K+-ATPase is the receptor for cardiac glycosides, a class of drugs used to treat congestive heart failure and arrhythymias. Binding of these compounds to the enzyme is antagonized by K+ ions suggesting that the binding sites for these ligands may either overlap or that binding of one may effect the other. Thus, defining the binding site for this class of drugs may help in understanding how Na+/K+-ATPase transports cations. An approach for defining the cardiac glycoside binding site is to use site-directed mutagenesis coupled with expression and selection systems. In addition, these techniques can be used to determine the role of specific amino acid residues in catalytic functions of the enzyme such as cation binding. Utilizing this approach we have identified amino acid residues which act as determinants of ouabain sensitivity as well as investigated the role specific transmembrane amino acids play in the catalytic activity of the enzyme. A functional approach is also being developed to determine if a naturally occurring ligand for Na+/K+-ATPase exists and whether it is physiologically significant.

Amino Acid Residues Involved in Ouabain Sensitivity and Cation Binding

The Na, K-ATPase, which is found in the cells of all higher eukaryotes, utilizes ATP to transport Na+ and K+ across the cell membrane. For every three sodium ions transported out of the cell two potassium ions are transported in. The enzyme is composed of two subunits, a larger a subunit, which is thought to contain most of the catalytic sites, and a smaller a subunit which is required for the proper processing and maturation of the enzyme. Three isoforms exist for the a subunit (α1, α2 and α3) and two isoforms exist for the β subunit (β1 and β2) in mammalian cells (Lingrel et al., 1990; Sweadner, 1989; Takeyasu et al, 1989). An additional isoform, β3, exists in Xenopous (Good et al., 1990). The a1 isoform is found in all cells while α2 is the primary isoform found in skeletal muscle, but is also present in the heart and nervous system. Expression of the α3 isoform is limited to the heart and nervous system (Orlowski and Lingrel, 1988). The cDNAs and genes corresponding to these isoforms have been isolated and the mechanisms responsible for their differential expression are being investigated (Lingrel et al., 1990). The Na,K-ATPase is a member of the P-type family of ATPases, which also include the Ca-ATPases and the H,K-ATPases. These transport proteins are similar in structure and have in common an aspart/l phosphate intermediate during their catalytic cycle. Site-directed mutagenesis and expression (MacLennan, 1990; Clarke et al., 1990; Vilsen and Andersen, 1992; Andersen and Vilsen, 1992) studies have been carried out to define cation binding sites in the Ca-ATPase but, less information is available using this approach with the Na.K-ATPase. The Na,K- ATPase differs from the other P-type ATPases in that it is sensitive to cardiac glycosides, a class of drugs which are used in the treatment of congestive heart failure and certain arrhythmias. The sensitivity of the enzyme to these drugs provides a useful tool for investigating structure-function relationships. In this report we describe progress made toward identifying potential cation binding sites as well as amino acid residues that are involved in determining cardiac glycoside sensitivity.