MOLECULAR BASIS OF ADRENERGIC RECEPTOR FUNCTION 
Brian K. Kobilka, M.D., Assistant Investigator 
Adrenergic receptors play an important role in the 
control of cardiovascular function by the central 
nervous system. The application of cloning, expres- 
sion, and mutagenesis techniques has led to remark- 
able progress in the field of adrenergic receptor biol- 
ogy. Much has been learned about the functional 
domains responsible for ligand binding, G protein 
activation, and receptor desensitization. Research in 
Dr. Kobilka's laboratory addresses several areas of 
adrenergic receptor biology that remain poorly 
understood. 
Receptor Structure 
Dr. Kobilka and his colleagues have attempted to 
gain insight into the three-dimensional structure of 
adrenergic receptors by identifying intramolecular 
interactions within the receptor protein. Chimeric 
a2/i32 receptors were constructed to locate comple- 
mentary mutations that restore functional proper- 
ties to mutationally inactivated receptors. The effect 
of mutations on receptor function may result either 
from a direct modification of a functional domain or 
from a more general effect on the three-dimensional 
structure of the receptor. The structural alterations 
are likely to occur when the mutated domain is in 
close proximity to another domain of the receptor, 
particularly if the chemical or physical properties of 
the mutated amino acid are essential for maintaining 
the three-dimensional architecture of the protein. 
Dr. Sankuratri Suryanarayana has taken advantage 
of these principles to study the structure of adrener- 
gic receptors. When Asn^'^ of the receptor is mu- 
tated to Phe ~^ F) ' the amino acid found in the 
homologous location of the a2 receptor, it is no 
longer functional and is retained in the endoplasmic 
reticulum. Inserting a sequence from the first and 
second hydrophobic domains of the a2 receptor into 
182N -»■ F restores the ability to bind ligands; this 
chimeric receptor is localized to the plasma mem- 
brane. The converse mutation in the a2 receptor 
(a2F ^ N) results in diminished agonist affinity. 
This abnormality can be complemented by inserting 
a sequence from the first hydrophobic segment of 
the receptor into 0:2? ~^ N. These results are con- 
sistent with a structural model in which Asn^'^ of 
the (82 receptor and Phe^'^ of the a2 receptor form 
important structural interactions with the first hy- 
drophobic segment. 
The arrangement of transmembrane segments 
predicted by these experiments is similar to that 
found in bacteriorhodopsin. This approach should 
facilitate the formulation and testing of more- 
accurate models for this class of membrane pro- 
teins. (This work was funded by a grant from the 
National Institutes of Health.) 
Receptor Biosynthesis 
Dr. Kobilka's group has used a cell-free expres- 
sion system to study the process by which receptors 
are translated, translocated into the endoplasmic re- 
ticulum membrane, and folded into a functional 
protein. The ^2 receptor is a type Illb membrane 
protein having multiple membrane-spanning do- 
mains with an extracellular amino terminus, but 
lacking a cleavable signal sequence. Dr. Xiaoming 
Guan has determined that the amino terminus and 
first hydrophobic segment of the ,82 receptor are in- 
efficiently translocated, resulting in a nonfunctional 
receptor. The addition of a cleavable signal se- 
quence to the amino terminus of the receptor 
greatly enhances translocation of the amino ter- 
minus and results in the production of more func- 
tional receptor protein. The increase in expression 
of functional protein afforded by the signal se- 
quence would not be expected to have physiologic 
significance, yet it remains to be explained why this 
class of membrane proteins has evolved with a less- 
efficient mechanism for translocation. Following 
translocation of the first hydrophobic domain, sub- 
sequent pairs of hydrophobic domains are inserted 
efficiently, and their insertion is not dependent on 
translocation of preceding domains. 
Post-transcriptional Regulation 
of the 182-Adrenergic Receptor 
The transcripts for the 182 receptor from all species 
studied thus far contain a small open reading frame 
(SORF) 5' to the coding sequence of the receptor. 
The peptide encoded by the SORF is well conserved 
across species. Dr. Tony Parola has obtained evi- 
dence that even though the SORF has a poor Kozak 
consensus sequence, it is efficiently translated in 
both a cell-free expression system and cultured 
cells. Translation of the peptide is linked to reduced 
synthesis of 182-receptor protein, because mutating 
the codon for the initiator methionine of the SORF 
leads to increased 182-receptor expression. 
Dr. Parola has observed that a synthetic peptide 
representing the product of the SORF is a potent 
inhibitor of translation in an in vitro expression sys- 
tem, suggesting that the product of the SORF nega- 
tively regulates 182-receptor synthesis. However, the 
CELL BIOLOGY AND REGULATION 8 1 
