To facilitate functional and mechanistic studies of receptor-G protein interactions, [corrected] the human beta 2-adrenergic receptor (h beta-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h beta-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h beta-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h beta-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by beta-adrenergic receptor agonists was achieved in cells coexpressing h beta-AR and a mammalian G protein (Gs) alpha subunit-demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.

The pleckstrin homology (PH) domain is a newly recognized protein module believed to play an important role in signal transduction. While the tertiary structures of several PH domains have been determined, some cocomplexed with ligands, the function of this domain remains elusive. In this report, the PH domain located in the N terminus of human phospholipase C-␦1 (PLC␦1) was found to regulate enzyme activity. The hydrolysis of phosphatidylinositol (PI) was stimulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in a dose-dependent manner with an EC 50 ‫؍‬ 1 M (0.3 mol%), up to 9-fold higher when 5 M (1.5 mol%) of PIP 2 was incorporated into the PI/phosphatidylserine (PS)/phosphatidylcholine (PC) vesicles (30 M of PI with a molar ratio of PI:PS:PC ‫؍‬ 1:5:5). Stimulation was specific for PIP 2 , since other anionic phospholipids including phosphatidylinositol 4-phosphate had no stimulatory effect. PIP 2 -mediated stimulation was, however, inhibited by inositol 1,4,5-triphosphate (IP 3 ) in a dose-dependent manner, suggesting a modulatory role for this inositol. When a nested set of PH domain deletions up to 70 amino acids from the N terminus of PLC␦1 were constructed, the deletion mutant enzymes all catalyzed the hydrolysis of the micelle forms of PI and PIP 2 with specific activities comparable with those of the wild type enzyme. However, the stimulatory effect of PIP 2 was greatly diminished when more than 20 amino acid residues were deleted from the N terminus. To identify the specific residues involved in PIP 2 -mediated enzyme activation, amino acids with functional side chains between residues 20 and 40 were individually changed to glycine. While all these mutations had little effect on the ability of the enzyme to catalyze the hydrolysis of PI or PIP 2 micelles, the catalytic activity of mutants K24G, K30G, K32G, R38G, or W36G was markedly unresponsive to PIP 2 . Analysis of PIP 2 -stimulated PI hydrolysis by a dual substrate binding model of catalysis revealed that the micellar dissociation constant (K s ) of PLC␦1 for the PI/ PS/PC vesicles was reduced from 558 M to 53 M, and the interfacial Michaelis constant (K m ) was reduced from 0.21 to 0.06 by PIP 2 . The maximum rate of PI hydrolysis (V max ) was not affected by PIP 2 . These results demonstrate that a major function of the PH domain of PLC␦1 is to modulate enzyme activity. Further, our results identify PIP 2 as a functional ligand for a PH domain and suggest a general mechanism for the regulation of other proteins by PIP 2 .

Pharmacologic, biochemical, and genetic analyses have demonstrated the existence of multiple a2-adrenergic receptor (a2AR) subtypes. We have cloned a human a2AR by using the polymerase chain reaction with oligonucleotide primers homologous to conserved regions of the previously cloned a2ARs, the genes for which are located on human chromosomes 4 (C4) and 10 (C10). The deduced amino acid sequence encodes a protein of 450 amino acids whose putative topology is similar to that of the family of guanine nucleotidebinding protein-coupled receptors, but whose structure most closely resembles that of the a2ARs. (13,14). In addition, our laboratory has shown that a probe made from the human platelet a2AR gene recognizes, at moderately high stringency, three different genes by Southern blot analysis of DNA from somatic cell hybrids (1). Each ofthese genes localizes to a different human chromosome: 2, 4, or 10 (a2C2, a2C4, and a2C10). The human platelet a2AR gene is located on chromosome 10. The gene for a second a2AR, which has been cloned from a human kidney cDNA library, is located on chromosome 4. We now report the cloning of a human a2AR whose gene is located on chromosome 2.tt The pharmacological properties of this receptor as well as its tissue distribution by Northern blot analysis suggest that the heterogeneity of this set of receptors may extend beyond the three pharmacologically defined subtypes. MATERIALS AND METHODSPolymerase Chain Reaction (PCR) Cloning. Oligonucleotide primers (primer A, 5'-GTGCAAGCTTGCACCTCGTC-GATCGTGCATCTGTGNGC-3'; primer B, 5'-CCCAAA-GAGCTCAGCCAGCACAAAGGTGAAGCG-3') corresponding to identified a2AR consensus sequences were synthesized on an Applied Biosystems 380 B DNA synthesizer and purified on a 16% (wt/vol) denaturing polyacrylamide gel or by HPLC using an Applied Biosystems Aquapore RP-300 column and a 5-95% acetonitrile gradient (15). The primers were designed with the restriction endonuclease linkers HindIII and Sac I at the 5' ends to facilitate subcloning. Sheared human genomic DNA (5 ,ug) tTThe sequence reported in this paper has been deposited in the GenBank data base (accession no. M34041). 5094The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.