ADRENERGIC RECEPTORS 
Robert J. Lefkowitz, M.D., Investigator 
The major themes of research in this laboratory 
are the elucidation of the molecular properties and 
regulatory influences that characterize the receptor 
binding sites for catecholamines such as epineph- 
rine (adrenaline) and norepinephrine (noradrena- 
line). These receptors can be categorized into two 
classes, termed a- and P-adrenergic receptors, 
which mediate the effects of catecholamines and re- 
lated drugs on a wide variety of physiological pro- 
cesses. 
I. (i-Adrenergic Receptors. 
A. Molecular properties. The laboratory continued 
to use techniques of site-directed mutagenesis and 
construction of chimeric receptors to delineate the 
structural basis of (3-adrenergic receptor function. 
Studies with chimeric P^- and -adrenergic receptors 
indicated that multiple membrane-spanning do- 
mains contribute to the binding specificity of the 
P-receptors. The fourth, sixth, and seventh trans- 
membrane helices appeared to be particularly im- 
portant. In contrast, the cytoplasmic domains, espe- 
cially the two ends of the third cytoplasmic loop 
found closest to the plasma membrane, appear to 
be important for determining specificity of coupling 
to G proteins. The most proximal region of the car- 
boxyl-terminal tail may contribute as well. 
The involvement of primarily membrane-span- 
ning domains in contributing to the ligand-binding 
site was confirmed by biochemical studies in which 
photoafifinity and affinity-labeling reagents were co- 
valently inserted into the ligand-binding site of ei- 
ther the P- or the a^-adrenergic receptors. After ap- 
propriate digestion by chemical or enzymatic 
means, the peptides to which the ligands were co- 
valently attached could be determined. These stud- 
ies indicated labeling of the second transmem- 
brane-spanning region by a covalent antagonist in 
the P2-adrenergic receptor and labeling of the 
fourth membrane-spanning domain by both the ag- 
onist and antagonist affinity reagents for the a^-ad- 
renergic receptors. 
The laboratory determined that a highly con- 
served cysteine found in the proximal portion of 
the carboxyl-terminal cytoplasmic tail of the p^-ad- 
renergic receptor is palmitoylated. This residue 343 
in the human p^-receptor is homologous with a cys- 
teine residue in rhodopsin, which also appears to 
be palmitoylated. In the case of the P-receptor, mu- 
tagenesis of this cysteine leads to complete loss of 
the palmitoylation, as well as partial impairment of 
receptor coupling. Although the exact significance 
of this palmitoylation is unknown, palmitate in this 
position of the receptor may serve to attach the 
cytoplasmic carboxyl-terminal tail of the receptor to 
the plasma membrane, thus creating a fourth 
cytoplasmic loop. 
B. Physiological regulation of ^adrenergic recep- 
tors. Investigation of phosphorylation of the P-ad- 
renergic receptor by the P-adrenergic receptor ki- 
nase (PARK) during homologous desensitization 
has been continued. Sufficient quantities of the ki- 
nase to obtain several limited stretches of protein 
sequence were purified, and oligonucleotide 
probes were designed and used to screen a bovine 
brain cDNA library. Full-length clones for the PARK 
were obtained. The deduced sequence indicates a 
protein of 686 amino acids with a calculated molec- 
ular weight of just under 80,000. There is a cen- 
trally located classic protein kinase catalytic do- 
main. This domain bears homology to all other 
protein kinase catalytic domains but is most similar 
(—33% sequence identity) to that of the cyclic nu- 
cleotide-dependent protein kinases and the C ki- 
nase family. The catalytic domain is flanked by two 
equal-sized domains that bear no homology to any 
currently sequenced protein. Messenger RNA for 
PARK is most abundant in brain, spleen, and heart, 
which are among the most heavily sympathetically 
innervated tissues. Southern blots reveal multiple 
bands hybridizing PARK probes with varying strin- 
gencies and intensities, suggesting the existence of 
a multigene family. Thus PARK appears to be the 
first sequenced member of a new gene family that 
may be of broad regulatory significance. 
Techniques of site mutagenesis were used to cre- 
ate site mutants lacking either the cAMP-dependent 
protein kinase phosphorylation sites or the pre- 
sumed sites of pARK phosphorylation at the car- 
boxyl terminus, or both sets of sites. All of these 
mutants activated adenylate cyclase and bound 
ligands normally. However, all showed decreased 
agonist-promoted phosphorylation and decreased 
agonist-promoted desensitization under appropri- 
ate experimental conditions. Work with these mu- 
tants has provided compeUing evidence that phos- 
phorylation of the receptor by several distinct 
protein kinases provides the molecular mechanism 
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