(Received for publication, January 23, 1995 )
From the
The binding of glucagon to its hepatic receptor triggers a
G-protein-mediated signal that ultimately leads to an increase in
hepatic glucose production (gluconeogenesis) and glycogen breakdown
(glycogenolysis). In order to elucidate the structural domain(s) of the
human glucagon receptor (hGR) involved in the selective binding of
glucagon, a series of chimeras was constructed in which various domains
of the hGR were replaced by homologous regions from the receptor for
the glucoincretin hormone, glucagon-like peptide I (GLP-IR). hGR and
GLP-IR are quite similar (47% amino acid identity) yet have readily
distinguishable ligand binding characteristics; glucagon binds to the
recombinant hGR expressed in COS-7 cells with a K that is 1000-fold lower than the K
for glucagon binding to GLP-IR. In the present study,
chimeric receptors were transiently expressed in COS-7 cells and
analyzed for glucagon binding. Expression of each receptor chimera was
confirmed by immunofluorescence staining using a hGR-specific
monoclonal antibody. This report identifies several non-contiguous
domains of the hGR that are important for high affinity glucagon
binding. Most notable are the membrane-proximal half of the
amino-terminal extension, the first extracellular loop, and the third,
fourth, and sixth transmembrane domains.
Glucagon is a 29-amino acid polypeptide hormone that is secreted
by the A cells of the islets of Langerhans and plays an important role
in the regulation of nutrient homeostasis(1) . The first step
in glucagon action is the binding of the hormone to the glucagon
receptor (GR) ()on the surface of liver cells (2) .
Upon glucagon binding, the receptor interacts with the
membrane-associated G-protein subunit G
to ultimately
stimulate adenylyl cyclase activity, leading to an increase in
intracellular cAMP. Elevation of this second messenger ultimately leads
to an increase in gluconeogenesis and glycogenolysis(3) .
The glucoincretin hormone known as glucagon-like peptide-I (GLP-I) is secreted in the gut and potentiates the glucose-induced secretion of insulin by the islets of Langerhans(4, 5) . Like glucagon, GLP-I exerts its effects by binding to its cognate receptor generating a rise in intracellular cAMP concentration(6) . Both hormones are of considerable importance in the control of blood glucose homeostasis.
The cDNAs encoding both the human glucagon receptor
(hGR) and the human islet GLP-I receptor (hGLP-IR) have been recently
cloned(6, 7) . Both polypeptides show sequence
similarity to a well established family of receptors with
seven-transmembrane domains that are known to couple to intracellular
effectors through heterotrimeric guanine nucleotide binding proteins (G
proteins)(8) . All members of this family of G-protein coupled
receptors (GPCRs) share the same basic structure and membrane topology.
Within the large family of GPCRs are grouped subfamilies based on
primary sequence homology(9) . The glucagon and GLP-I peptide
receptors are grouped into a subfamily that includes the receptors for
secretin, calcitonin, vasoactive intestinal peptide, parathyroid
hormone, and growth hormone-releasing factor(9) . The hGR and
hGLP-IR share 47% amino acid identity and 66% similarity, with the
transmembrane segments having the highest degree of similarity (see Fig. 1). Despite this homology, glucagon binds to the hGR with a K approximately 1000-fold lower than to
the hGLP-IR.
Figure 1: Conservation of primary structure: hGR versus GLP-I receptor. This ``serpentine'' diagram indicates the predicted transmembrane domains as well as the extra- and intracellular loops of the human glucagon receptor. The arrangement depicted here is based on what is known of the family of GPCRs(9) . Residues with a whitebackground indicate amino acid identity between hGR and hGLP-IR; white on black residues represent non-conserved amino acid changes; and residues with a gray background indicate conservative evolutionary substitutions. Amino acids are numbered (as shown and throughout the text) starting with the initiator methionine and proceeding toward the carboxyl terminus. Roman numerals refer to the numbering of TMDs as referred to in the text. Membrane-spanning regions are as predicted previously(7) .
Despite a large body of information regarding the
ligand binding and signaling characteristics of GPCRs in general, there
has been very little structure-function analysis reported about the
glucagon-secretin subfamily. In order to understand the nature of the
interaction of glucagon with its receptor, we constructed a number of
hGRhGLP-IR chimeras in which the various domains of the hGR were
sequentially replaced by homologous domains from hGLP-IR. Since both
receptors are highly conserved throughout their entire length, it was
possible to swap domains without introducing any gaps or deletions in
the primary amino acid sequence. We report here the identification of
several, non-continuous domains that are required for high affinity
glucagon binding.
Restriction endonucleases were from
Boehringer Mannheim. Polymerase chain reaction reagents were from
Invitrogen Corp. and New England Biolabs (Vent polymerase). I-Labeled glucagon was obtained from DuPont, and
unlabeled glucagon was purchased from Sigma. COS-7 cells were obtained
from ATCC.
The wild type, mutant, and chimeric receptors were expressed transiently by DEAE-mediated transfection of COS-7 cells(13) .
Figure 2:
Schematic representation of the
hGRhGLP-IR chimeras. Amino acids indicated below each chimera
refer to the residues of the hGR present in each construct. Dark
line, hGR; gray line, hGLP-IR. Numbering corresponds to
that used in Fig. 1.
Figure 3:
Competition curves of glucagon binding to
chimeric receptors. I-Labeled glucagon was used as a
radioligand tracer to analyze the binding of glucagon to various
chimeras present in transfected COS-7 cells. Shown is percent maximum
binding versus nM unlabeled glucagon for each
construct. A, competition curves comparing hGR (
), GLP-IR
(
), CH-1 (
), CH-2 (
), CH-3 (
), CH-4
(
), and CH-5 (
). B, competition curves comparing
hGR (
), GLP-IR (
]), CH-6 (
), and CH-7 (
). C, competition curves comparing hGR (
), GLP-IR (
),
CH-8 (
), CH-9 (
), and CH-10 (
). Each point is the average of triplicate measures. Standard error in each case
is within ±6%.
Replacement of more than half of the amino-terminal extension (chimera CH-1) with the homologous region from hGLP-IR had little or no effect on ligand binding. However, replacement of the entire amino-terminal extension and the first intracellular loop, including transmembrane domains (TMDs) I and II (chimera CH-2) abolishes high affinity binding (Fig. 3A). Furthermore, chimera CH-3, in which the first intracellular loop plus TMDs I and II are replaced, yields a construct with a high binding affinity. Taken together, binding data from CH-1, CH-2, and CH-3 suggest that the membrane-proximal region of the amino-terminal extension is in fact important for glucagon binding or recognition, whereas the membrane-distal half of the amino-terminal extension, the first intracellular loop, and TMD I and II are not involved in a major way.
Substitution of the first extracellular loop plus TMDs III and IV (chimera CH-4) yields a construct that has no high affinity glucagon binding activity (Fig. 3A). However, chimera CH-5, in which the majority of the second intracellular loop was replaced, binds glucagon with high affinity. Data from CH-4 and CH-5 together suggest that the first extracellular loop and TMDs III and IV are involved in glucagon-specific binding, but the second intracellular loop is not.
In order to determine whether the domains surrounding the second extracellular loop are important, CH-6 and CH-7 were constructed. CH-6, in which TMD V plus the third intracellular loop have been replaced, binds glucagon with high affinity, as does CH-7 in which the second extracellular loop has also been switched (Fig. 3B). These results indicate that the second extracellular loop, TMD V, and the third intracellular loop are not involved in high affinity glucagon binding.
CH-8, CH-9, and CH-10 were designed to address the role of the carboxyl-terminal region of the receptor in ligand binding. Chimera CH-8 is able to bind glucagon with high affinity, which indicates that TMD VII plus the entire intracellular carboxyl-terminal tail is not involved in ligand selection (Fig. 3C). CH-10, in which the third extracellular loop has effectively been converted into GLP-IR, binds glucagon but with somewhat reduced affinity relative to the wild-type receptor, even though this chimera differs in only two adjacent amino acids: Ser-379 to Phe and Ala-380 to Ile. CH-9, however, binds glucagon with an affinity comparable with that of the GLP-IR. These data indicate that TMD VI is important for glucagon binding.
In summary, the regions which are not involved in the selective binding of glucagon include the membrane-distal half of the amino-terminal extension, all three intracellular loops, the second extracellular loop, TMDs I, II, V, and VII, and the entire carboxyl-terminal tail. The first and third extracellular loops appear to be involved, as do TMDs III, IV, and VI, as well as the membrane-proximal half of the amino-terminal extension. These results are summarized under ``Discussion'' and in Fig. 5.
Figure 5: Regions of the hGR implicated in high affinity glucagon-specific binding. The composite diagram indicates regions of the receptor that are involved in the specificity of glucagon binding. Whitecircles, individual residues that are not involved in the selective binding of glucagon or amino acid residues that are identical between the two receptors. Blackcircles, residues that have been shown to be required for glucagon binding or non-identical residues between the two receptors.
Figure 4: Cellular distribution of the hGR chimeras in transfected COS-7 cells. Transfected viable COS-7 cells were immunolabeled with a fluorescent tag (fluorescein isothiocyanate). Leftpanel, fluorescence staining; rightpanel, phase-contrast micrographs. In control experiments using only preimmune sera, no staining could be detected. A, hGR; B, CH-2; C, CH-4; D, CH-9; E, CH-10; F, hGLP-IR.
The construction of chimeric receptors is a useful starting point for identifying structural domains involved in the selective binding of a ligand to its receptor. The strategy in such experiments is to exchange homologous domains between two similar, yet functionally distinct receptors and correlate gain or loss of function with the exchanged domain. This approach eliminates any gross structural alterations such as altered folding or membrane insertion that can occur with other methods such as substitution or deletion mutagenesis. However, while the design of chimeras is a useful method for identifying amino acid residues involved in ligand-specific selectivity, this approach does not necessarily identify domains that may be important for ligand binding but are common to both receptors. A composite diagram indicating the regions of the hGR that are involved in the selective binding of glucagon is shown in Fig. 5. The residues in Fig. 5shown as ``not involved'' in high affinity glucagon-specific binding are determined by two criteria: 1) chimera data and 2) sequence identity between the hGR and GLP-IR.
The general picture that has emerged of the peptide ligand binding site on GPCRs implicates the extracellular domains, and less often, a few select transmembrane segments. This is true even of very short peptide ligands such as the tripeptide fMet-Leu-Phe, which binds to the N-formyl peptide receptor via multiple, discontinuous regions, especially in the extracellular domains and near the top of the transmembrane regions(14) . Our structure-function analysis of the hGR indicates that it conforms well to this paradigm. Most GPCRs that bind large peptides utilize residues within the amino-terminal extension(9, 15) . Our findings are consistent with this idea. Specifically, we find that residues between Ser-80 and Gln-142 are necessary for glucagon binding specificity. In addition, a recent study by Carruthers et al.(16) demonstrated by site-directed mutagenesis that a highly conserved aspartic acid residue located within the amino-terminal extension of the rat GR (corresponding to Asp-63 in Fig. 1) is crucial for ligand binding. This conserved aspartic acid residue is probably of general structural importance for the binding of a number of peptide ligands within the family of GPCRs and is not involved in ligand selectivity.
Huang et al.(17) have demonstrated that residues lying within TMD II and VII of the neurokinin-1 receptor are important for the specificity of binding of its peptide ligand, substance P; in contrast, we find that residues within TMD II and TMD VII do not appear to be involved in glucagon-specific binding. A two-amino acid change within extracellular loop 3 of the hGR (CH-11) leads to a slight but significant decrease in glucagon-specific binding, similar to the interleukin-8 receptor, which utilizes two residues within its third extracellular loop in the specific binding of interleukin-8(18) .
Early biochemical measurements of the site(s) of glucagon interaction with its hepatic receptor demonstrated that both the glucagon binding function of the receptor as well as the region functionally coupled to adenylyl cyclase are contained within a 21-kDa peptide fragment(19) . These experiments involved the proteolytic treatment of the receptor following covalent cross-linking with monoiodoglucagon. A subsection of the receptor (approximately the carboxyl-terminal two-thirds of the receptor, including all of the hydrophobic membrane binding regions) was identified as the fragment that binds glucagon. This conclusion is consistent with the data presented here.
More recently, effort has been directed toward a
structural analysis of glucagon, specifically the residues involved in
receptor binding (20, 21, 22) . Hjorth et
al.(20) studied chimeric glucagonGLP-I peptide
hormones' abilities to bind to the rat GR and GLP-IR. They
determined that the epitope involved in the selective recognition of
the GR is located near the amino terminus of glucagon, and the GLP-I
selective recognition epitope is located in the far carboxyl-terminal
end of GLP-I. While such analyses do not predict the receptor domains
involved in binding, they provide some information on receptor-ligand
interaction. A deeper understanding requires the complementary analysis
of the receptor itself. Our future effort will be directed toward a
detailed structural characterization of the residues of the receptor
involved in glucagon recognition and specific contact.