ACTH Receptor

ACTH Receptor F. Shawn Galin and J. Edwin Blalock* Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA * corresponding author tel: 205-934-6439, fax: 205-934-1446, e-mail: [email protected] DOI: 10.1006/rwcy.2000.23007.

SUMMARY The ACTH receptor is a member of the melanocortin receptor group, five members of which have been described. Melanocortin receptor 2 is used by adrenal cells to interact with ACTH. Although ACTH has numerous effects on immune cells that occur through an ACTH receptor, it is unknown at present which member of the melanocortin receptor family is used.

BACKGROUND

Discovery The field of neuroimmunoendocrinology emerged based on the finding that cells of the neuroendocrine and immune systems could communicate with one another. The biochemical rationale for this bidirectional communication between the neuroendocrine and immune systems lies in the fact that these two systems share ligands and receptors. The first neuroendocrine hormone shown to be produced by immune cells was the pro-opiomelanocortin (POMC)derived hormone corticotropin, or ACTH (Blalock and Smith, 1980). At that time, this finding was puzzling since the only known receptors of the pituitary-derived counterpart were thought to reside exclusively in the adrenal gland. However, this notion was soon dispelled after it was shown that ACTHbinding sites do reside on cells of the immune system. The first evidence to imply that ACTH-binding sites resided on cells of the adrenal gland was the finding that treatment of adrenal tissue with exogenous ACTH led to an increase in glucocorticoid production which could be blocked by antibody to ACTH (Taunton et al., 1967). When it was shown that ACTH had biological effects on lymphocytes,

much effort was expended to determine whether ACTH receptors resided on cells of the immune system. One of the first pieces of evidence that ACTH receptors were expressed on lymphocytes was shown through binding studies using radiolabeled ACTH as a probe. In these studies both a specific high-affinity binding site and a low-affinity binding site were found on the surface of mouse spleen cells (Johnson et al., 1982). These values corresponded to those seen on mouse adrenal cells. More recently, using an iodinated ACTH analog, a number of high-affinity binding sites were detected on purified B and T cell populations (Clarke and Bost, 1989). Since both the adrenal and the lymphocyte binding sites are comparable with regard to affinities, it would seem that the receptors are structurally and functionally related. The number of receptors expressed on lymphocytes is not static since mitogen stimulation can markedly increase the number of receptor sites on both B and T cells.

Alternative names The ACTH receptor belongs to a family of receptors known as the melanocortin (MC) receptors. The melanocortins include ACTH and the melanocytestimulating hormones (MSH). The MSHs are appropriately named for their ability to act on melanocytes and alter pigmentation. The sequence for the MSHs resides within ACTH itself. The sequence for -MSH corresponds to the first 13 amino acids of ACTH, whereas -MSH is identical to ACTH residues 4±10. Considering this, it is not surprising that ACTH can act on melanocytes. Furthermore, it is not unusual for a rise in MSH activity to accompany a rise in steroidogenic activity (for review see Inouye and Otsuka, 1987).

2236 F. Shawn Galin and J. Edwin Blalock The MSH peptides arise from differential processing of the precursor POMC in the pituitary gland. In the anterior lobe, corticotrophs process POMC into ACTH and -endorphin whereas the melanotrophs yield -MSH and corticotropin-like intermediate peptide or CLIP. Aside from their classical functions, melanocortins have been shown to have other effects on cognitive function (DeWeid and Jollies, 1982), cardiovascular regulation (Gruber and Callahan, 1989), thermoregulation (Lipton et al., 1981), and appetite (Mountjoy and Wong, 1997).

Structure To date, there are five known MC receptors, termed MC1-R through MC5-R. They are all members of the G protein-linked seven membrane spanning domain superfamily. They are approximately 300 (Cammas et al., 1995) to 360 (Gantz et al., 1993a) amino acids in length. Since the MSH receptor was the first to be cloned, it received the title MC1-R. The adrenal ACTH receptor, which was next, is termed MC2-R. MC3-R, MC4-R, and MC5-R are all in numerical order from when they were discovered. The highest homology within the MC receptors lies in the transmembrane-spanning domains (TM) themselves. They contain the highly conserved Asp-Arg-Tyr motif in TM region 3 and the second intracellular loop seen within the seven membrane spanning domain receptor superfamily (Probst et al., 1992). They differ with other members of the superfamily in that they lack highly conserved structural residues such as the prolines which reside in the fourth and fifth TM domains and the cysteines in TM-1 and TM-2 (Probst et al., 1992; Tatro, 1996).

Main activities and pathophysiological roles The classical function attributed to the adrenal ACTH receptor is to stimulate steroidogenesis in response to a physical stressor. Thus, the receptor plays a key role in the hypothalamic-pituitary-adrenal axis. Briefly, in response to stress, there is an increase in the secretion of corticotropin-releasing hormone (CRH) from the hypothalamus, which leads to an increase in synthesis, and secretion of ACTH by corticotrophs in the pituitary gland. The pituitaryderived ACTH then, in turn, acts on cells of the adrenal cortex to stimulate the release of glucocorticoids. This is achieved by both the upregulation of enzymes involved in steroidogenesis as well as

the activation of the conversion of cholesterol to pregnenolone, which is ultimately converted to corticosterone or cortisol in the rodent and in humans, respectively. There is negative feedback regulation of this axis where increasing levels of glucocorticoids feedback to the levels of both the hypothalamus and the pituitary to inhibit the release of CRH and ACTH, respectively. The lymphocyte ACTH receptor has a variety of responses attributed to its modulation of immune function. Corticotropin has been shown to have direct effects on lymphocytes such as mitogen-stimulated proliferation (Alvarez-Mon et al., 1985) and antibody responses to both T cell-dependent and -independent antigens (Johnson et al., 1982). Interestingly, ACTH was shown to reverse the inhibitory effects of dexamethasone on lymphocyte proliferation, which would suggest that ACTH may serve to `protect' the immune response from the effects of transient or sustained rises in glucocorticoid levels which are known to have pronounced inhibitory effects on immune function (Pepper et al., 1993).

GENE

Accession numbers To date, five MC receptors have been described. Using the polymerase chain reaction, the cDNA from a human melanoma was used to clone and sequence the MC1-R (GenBank X65634) and the MC2-R (GenBank X65633) genes (Mountjoy et al., 1992). These MC receptor genes lacked introns, which is not unlike the genes encoding many other members of the G protein-linked seven membrane-spanning domain receptor superfamily. The genes for the third (GenBank L06155), fourth (GenBank L08603), and fifth (GenBank U08353) melanocortin receptor have also been cloned and sequenced (Gantz et al., 1993a,b; Fathi et al., 1995). The adrenal ACTH receptor has been identified as the MC2-R. This is consistent with a recent finding that the melanocortin 1, 3, 4, and 5 receptors do not have an epitope for ACTH outside of the sequence for -MSH (ACTH 1±13) (Schioth et al., 1997).

PROTEIN

Accession numbers GB299420

ACTH Receptor 2237

Sequence See Figure 1.

Relevant homologies and species differences There is 40±80% homology between receptor subtypes. Within a subtype there is 80±95% homology between different species.

Affinity for ligand(s) See Table 1.

Cell types and tissues expressing the receptor In addition to the classic site at the cortex of the adrenals (Table 1), ACTH receptors are also expressed in adipose tissue and in B cells and T cells within the immune system. Cell lines expressing the receptor include murine Y-1 and human H295 adrenocortical cells.

Regulation of receptor expression It has been shown that ACTH can upregulate the expression of the ACTH-binding site and receptor

transcripts several-fold in murine Y-1 and human H295 cells (Mountjoy et al., 1992). In lymphocytes, the levels of receptor expression can be increased upon mitogen stimulation. For example, lipopolysaccharide stimulation of B cells can result in a 2-fold increase in receptor expression, whereas T lymphocytes and thymocytes stimulated with concanavalin A have been shown to increase receptor expression by 3- and 1000-fold, respectively (Clarke and Bost, 1989). This increase in receptor expression and subsequent binding by ACTH has consequences on immune function. For example, ACTH has been shown to upregulate IL-6 and IL-4 production, which are cytokines associated with a TH2-type T cell response (Aebischer et al., 1994). Interestingly, lymphocyte-derived TGF can downregulate ACTH binding and block upregulation of ACTH-binding sites by ACTH itself (Rainey et al., 1989). Thus it seems that the ACTH receptor is subject to negative feedback regulation by TGF .

SIGNAL TRANSDUCTION

Cytoplasmic signaling cascades As previously mentioned, the ACTH receptors are G protein-coupled receptors. Binding of ACTH to its receptor in both adrenal and lymphoid cells results in an increase or rise in cyclic AMP (cAMP) and cGMP levels. In addition to the change in adenylate cyclase activity, the adrenal ACTH receptor is thought to be linked to steroidogenesis through a calcium flux in

Figure 1 Amino acid sequence for the ACTH receptor. 1 51 101 151 201 251

MKHIINSYEN KNKNLQAPMY TADDIIDSLF LTVIWTFCTG RSHTRKISTL CACYMSLFQV

INNTARNNSD FFICSLAISD VLSLLGSIFS TGITMVIFSH PRANMKGAIT NGMLIMCNAV

CPRVVLPEEI MLGSLYKILE LSVIAADRYI HVPTVITFTS LTILLGVFIF IDPFIYAFRS

FFTISIVGVL NILIILRNMG TIFHALRYHS LFPLMLVFIL CWAPFVLHVL PELPRDAFKK

ENLIVLLAVF YLKPRGSFET IVTMRRTVVV CLYVHMFLLA LMTFCPSNPY MIFC

Table 1 Ligand preference and tissue distribution of MC receptors Receptor

Tissue distribution

Specificity

Reference

MC1-R

Melanocytes, adipose tissue

-MSH > ACTH > -MSH

Mountjoy et al., 1992

MC2-R

Adrenal cortex

ACTH

Mountjoy et al., 1992

MC3-R

Brain, gut, placenta

-MSH = -MSH = ACTH

Gantz et al., 1993

MC4-R

Brain, sympathetic nervous system

-MSH = ACTH > -MSH

Gantz et al., 1993

MC5-R

Adipose tissue, skeletal muscle, pituitary

-MSH > ACTH > -MSH

Fathi et al., 1995

2238 F. Shawn Galin and J. Edwin Blalock these cells (Saez et al., 1981; Tremblay et al., 1991). In lymphocytes, the addition of ACTH was also found to increase calcium uptake in these in a time- and dose-dependent manner. The kinetics are similar to that seen in adrenal cells. Furthermore, this mechanism is blocked by calcium channel antagonists (Clarke et al., 1994). A rise in cytosolic free calcium is one of the first responses involved in lymphocyte activation. It has been shown that ACTH can modulate both B and T cell proliferation and function. Interestingly, it has been shown that ACTH alone does not alter the basal levels of cytosolic free calcium (Kavelaars et al., 1988; Clarke et al., 1994); however, this is not unlike that seen in adrenal cells (Iida et al., 1986). In lymphocytes, ACTH works in concert with mitogen or antigen stimulation to cause a calcium flux across the plasma membrane. The activity of ACTH seems to alter calcium levels through increasing calcium channel activity rather than the release of calcium through intracellular stores (Clarke, 1995).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors The activation of the ACTH receptor is a primary event in steroidogenesis, leading to alterations in circulating glucocorticoid levels. In fact, a mutation in the gene has been described in glucocorticoid deficiency syndrome (Slavotinek et al., 1998). This is characterized by extremely low cortisol levels in the face of high ACTH concentrations.

Human abnormalities A mutation was shown to lead to a new restriction endonuclease site with the ACTH receptor gene, allowing for detection without the need for DNA sequencing. Interestingly, glucocorticoid deficiency syndrome resulting from decreased ACTH receptor expression may not be confined exclusively to cells of the adrenal gland. Indeed, the ability to detect a form of glucocorticoid deficiency due to ACTH insensitivity syndrome by testing for the absence of high-affinity ACTH-binding sites on a patient's peripheral blood

mononuclear cells may be the first example (Smith et al., 1987; Yamamoto et al., 1995). Two mutations in the receptor gene have been shown to be associated with human ACTH hypersensitivity syndrome. Using PCR and nucleotide sequencing techniques, two base mutations were found. The first was cysteine 21 to arginine and the second, serine 247 to glycine. These regions corresponded to the first extramembranous N-terminal domain and the third extramembranous loop, respectively. Clinically, this led to normal cortisol levels in the absence of detectable ACTH levels and an increase in adrenocorticol sensitivity to ACTH (Hiroi et al., 1998).

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