Site-directed mutagenesis was used to examine the importance of five carboxyl-containing amino acids localized in the putative membrane-spanning regions of the Na,K-ATPase (i.e., E327, E778, D803, D807, and D925 of the rat alpha 2 isoform). The substitutions were introduced into a cDNA encoding a ouabain-resistant isoform (i.e., rat alpha 2* which was mutated to encode a ouabain-resistant isoform), and the effect of these substitutions on Na,K-ATPase function was assessed by screening the altered enzymes for their ability to confer ouabain resistance when expressed in otherwise ouabain-sensitive cells. The expression of the alpha isoform containing certain substitutions at positions 327 and 925 was able to confer ouabain resistance to HeLa cells while the expression of rat alpha 2* containing substitutions at positions 778, 803, and 807 was not. In particular, amino acids in each of these positions were substituted with leucine to evaluate the importance of the carboxyl-containing side chain. The ability of rat alpha 2* containing E327L and D925L to confer ouabain resistance to HeLa cells indicates that neither the negative charge nor the oxygen-containing side chain is absolutely essential for overall function in this position. In contrast, the inability of rat alpha 2* carrying E778L, D803L, and D807L to confer ouabain resistance suggests that the naturally occurring amino acid may be more critical structurally and/or functionally for the Na,K-ATPase. Other more conservative substitutions introduced to further characterize the role of particular amino acid side chains include E327D, E327Q, D803N, D803E, and D925N.(ABSTRACT TRUNCATED AT 250 WORDS)

The ␣ 2 -adrenergic receptor (␣ 2 AR) subtype ␣ 2 C10 undergoes rapid agonist-promoted desensitization which is due to phosphorylation of the receptor. One kinase that has been shown to phosphorylate ␣ 2 C10 in an agonistdependent manner is the ␤AR kinase (␤ARK), a member of the family of G protein-coupled receptor kinases (GRKs). In contrast, the ␣ 2 C4 subtype has not been observed to undergo agonist-promoted desensitization or phosphorylation by ␤ARK. However, the substrate specificities of the GRKs for phosphorylating ␣ 2 AR subtypes are not known. We considered that differential capacities of various GRKs to phosphorylate ␣ 2 C10 and ␣ 2 C4 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 ␣ 2 C10 or ␣ 2 C4 without or with the following GRKs: ␤ARK, ␤ARK2, GRK5, or GRK6. Intact cell phosphorylation studies were carried out by labeling cells with 32 P i , exposing some to agonist, and purifying the ␣ 2 AR by immunoprecipitation and SDS-polyacrylamide gel electrophoresis.␤ARK and ␤ARK2 were both found to phosphorylate ␣ 2 C10 to equal extents (>2-fold over that of the endogenous kinases). On the other hand, GRK5 and GRK6 did not phosphorylate ␣ 2 C10. In contrast to the findings with ␣ 2 C10, ␣ 2 C4 was not phosphorylated by any of these kinases. Functional studies carried out in transfected HEK293 cells expressing ␣ 2 C10 or ␣ 2 C4 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 ␣ 2 C10 by co-expression of ␤ARK but not GRK6, while ␣ 2 C4 failed to desensitize with co-expression of ␤ARK.These results indicate that short term agonist-promoted desensitization of ␣ 2 ARs by phosphorylation is dependent on both the receptor subtype and the expressed GRK isoform.During continuous activation by agonists, many G proteincoupled receptors display the property of desensitization, which is manifested as a waning of receptor function over time (1). The ␣ 2 adrenergic receptors (␣ 2 AR) 1 signal via multiple effectors, including the inhibition of adenylyl cyclase by coupling to the inhibitory G protein G i . With the ␣ 2A AR (also denoted as human ␣ 2 C10), this signaling has been shown to undergo such desensitization after brief periods of agonist exposure (2, 3). The primary mechanism of rapid desensitization of ␣ 2 C10 appears to be receptor phosphorylation, which ultimately leads to receptor-G protein uncoupling. Several lines of evidence implicate the ␤-adrenergic receptor kinase or a related G protein-coupled receptor kinase (GRK), as the kinase responsible for desensitization of ␣ 2 C10. Desensitization of ␣ 2 C10 occurs after seconds to minutes of agonist exposure, requires saturating concentrations of agonist, is inhibited by the ␤ARK inhibitor heparin, and is ablated in a mutant receptor lacking phosphoryl...

The alpha 2C2 adrenergic receptor contains a highly acidic stretch of amino acids (EDEAEEEEEEEEEEEE) within the third intracellular loop. To investigate the role of this region, we utilized site-directed mutagenesis to delete these 16 amino acids as well as to substitute them with glutamine, thereby conserving size but not charge. The wild-type and mutated alpha 2C2 receptors were permanently expressed in CHO cells. Neither substitution nor deletion of this region affected receptor expression, agonist or antagonist binding affinities, guanine nucleotide-sensitive formation of the high-affinity agonist-receptor-G protein complex, or functional coupling of the receptor to Gi. We considered that since alpha 2C2 agonist-promoted desensitization is due to phosphorylation by the beta-adrenergic receptor kinase (or a related kinase), that this region may be important for establishing the acidic mileau required by this kinase. Therefore, the consequences of 30 min of agonist preexposure on subsequent alpha 2C2-mediated inhibition of adenylyl cyclase and on high-affinity agonist binding were determined for the wild-type and these two mutants. The wild-type alpha 2C2 receptor underwent approximately 52% functional desensitization and a approximately 40% loss of high-affinity binding after such exposure. In contrast, deletion and substitution of this acidic stretch of amino acids ablated desensitization as assessed by both approaches. These results correlated with those obtained in whole cell phosphorylation experiments. Cells expressing each receptor were incubated with [32P]orthophosphate and exposed to agonist, and receptors were purified by immunoprecipitation. The deletion and the substitution mutant receptors underwent agonist-promoted phosphorylation at levels of only 44 +/- 5% and 50 +/- 15%, respectively, relative to wild-type alpha 2C2.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated the potential for protein kinase C (PKC) to phosphorylate and desensitize the ␣ 2A -adrenergic receptor (␣ 2A AR). In whole-cell phosphorylation studies, recombinantly expressed human ␣ 2A AR displayed an increase in phosphorylation after short-term exposure to 100 nM phorbol 12-myristate-13-acetate (PMA) that was blocked by preincubation with a PKC inhibitor. This increase in receptor phosphorylation over basal amounted to 172 Ϯ 40% in COS-7 cells and 201 Ϯ 40% in Chinese hamster ovary cells. In permanently transfected Chinese hamster fibroblast cells, PKC activation by brief exposure of the cells to PMA resulted in a marked desensitization of ␣ 2A AR function, amounting to a 68 Ϯ 4% decrease in the maximal agonist (UK14304)-stimulated intracellular calcium release. Such desensitization was blocked by the PKC inhibitor bisindolylmaleimide I and was not evoked by an inactive phorbol ester. The desensitization of this agonist response was not caused by PKC-mediated augmentation of G protein-coupled receptor kinase activity, because PMA-promoted desensitization of a mutated ␣ 2A AR that lacked G protein-coupled receptor kinase phosphorylation sites was identical to that of wild-type ␣ 2A AR. To test whether PKC phosphorylation is a mechanism by which ␣ 2A AR can be regulated by other receptors, the ␣ 1b AR was co-expressed with the ␣ 2A AR in Chinese hamster ovary cells. Upon selective activation of ␣ 1b AR, the function of ␣ 2A AR underwent a 53 Ϯ 5% desensitization. Thus, cellular events that result in PKC activation promote phosphorylation of the ␣ 2A AR and lead to substantial desensitization of receptor function. This heterologous regulation also represents a mechanism by which rapid crosstalk between the ␣ 2A AR and other receptors can occur.The ␣ 2 ARs regulate several effector systems including adenylyl cyclase, potassium channels, calcium channels, and inositol phosphate-mediated intracellular calcium release (Limbird, 1988;Liggett, 1996;Akerman et al. 1997;. Studies in intact organisms and cell culture systems have indicated that the function of G protein-coupled receptors, including ␣ 2 ARs, can be dynamically regulated under various physiologic and pathophysiologic conditions (Liggett and Lefkowitz, 1993;Liggett, 1997). We have recently delineated one pathway whereby persistent agonist activation results in a dampening of ␣ 2 AR signaling, termed desensitization, which is due to receptor phosphorylation by GRKs (Eason and Liggett, 1992;Liggett et al., 1992;Eason et al., 1995). GRK-mediated desensitization is evoked by agonist occupancy of the receptor, is independent of the generation of second messengers, and represents one mechanism of homologous desensitization of the ␣ 2 AR. The molecular basis of heterologous regulation of ␣ 2 AR has been largely unexplored. In the current study, we investigated the regulation of ␣ 2 AR signaling by PKC. This kinase was found to phosphorylate the ␣ 2A subtype, which resulted in a rapid desensitization of receptor function. These effect...

A prominent feature of long-term regulation of the alpha2A-adrenergic receptor (alpha2AAR) is a loss of cellular receptors over time (downregulation). The molecular determinants of downregulation were sought by targeting regions of the receptor involved in G protein coupling and phosphorylation. Mutated receptors, consisting of chimeric substitutions of analogous beta2-adrenergic receptor (beta2AR) and serotonin 5-hydroxytryptamine1A (5-HT1A) receptor sequence into the second intracellular loop (ICL2) (residues 113-149), the amino terminus (residues 218-235) and carboxy terminus (residues 355-371) of ICL3, and a deletion of the beta-adrenergic receptor kinase (betaARK) phosphorylation sites in the third intracellular loop (ICL3) (residues 293-304), were expressed in Chinese hamster ovary (CHO) cells. Wild-type alpha2AAR underwent 31% +/- 3% downregulation after 24 h of exposure to 100 microM epinephrine. Loss of downregulation was observed with some mutants, but this was not related to functional coupling to inhibitory or stimulatory guanine nucleotide regulatory binding proteins (Gi or GS) or to phosphorylation. Rather, any mutant with a substitution of the amino terminus of ICL3 (regardless of whether the substitution was with beta2AR or 5-HT1A sequence) resulted in upregulation. Studies with an inhibitor of protein synthesis indicated that the primary mechanism of downregulation of the alpha2AAR is agonist-promoted degradation of receptor protein which requires a destabilization sequence in the amino terminus of ICL3. Thus, in contrast to other G protein-coupled receptors, in which G protein coupling or phosphorylation are critical for long-term agonist regulation, the alpha2AAR has a specific structural domain distinct from these other functional regions that serves to direct agonist-promoted downregulation.